Compositions and methods for producing engineered immune cells

Abstract

The present disclosure provides compositions and methods for producing an engineered immune cell including a first nucleotide sequence encoding a T-cell receptor (TCR) and a second nucleotide sequence encoding a co-stimulatory receptor molecule. The present disclosure also provides methods for producing the engineered immune cells comprising a plurality of antigen specific T cells. The methods can be used to produce a therapeutically effective amount of antigen specific T cells within a short period of time. The compositions and methods provided herein can be used for the treatment of cancer.

Description

COMPOSITIONS AND METHODS FOR PRODUCING ENGINEERED IMMUNECEEESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 638,230, filed April 24, 2024, which application is incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Cancer is a leading cause of death worldwide, accounting for nearly one in six deaths globally. While cell-based immunotherapies have led to advances in treatment options for cancer, conventional engineered cell therapy manufacturing may require extensive time in cell culture, which is both expensive and can negatively affect the fitness of the engineered cells. The current short manufacturing protocols can often lead to low engineering efficiency. There is a need for efficient manufacturing methods that are both time and cost effective.SUMMARY OF THE INVENTION

[0003] Recognized herein is a need to improve the conventional engineered cell therapy manufacturing methods. The methods and compositions provided herein can improve the efficacy and reduce the time commitment for generating engineered immune cells.

[0004] Provided herein is an engineered immune cell comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; and a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous costimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed.

[0005] In some embodiments, the engineered immune cell further comprises an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell. In some embodiments, the exogenous TCR molecule binds to the exogenous peptide when presented in complex with the MHC.

[0006] Also provided herein is an engineered immune cell comprising a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule; and an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell, wherein the exogenous TCR molecule binds to the exogenous peptide when presented in complex with the MHC.

[0007] In some embodiments, (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous costimulatory receptor molecule is transiently expressed. In some embodiments, the exogenous co-stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC). In some embodiments, the activation of the exogenous co-stimulatory receptor molecule provides a co-stimulatory signal to the engineered immune cell.

[0008] In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. In some embodiments, the exogenous co- stimulatory receptor molecule or the surface protein comprises CD70 and / or CD80. In some embodiments, the exogenous co-stimulatory receptor molecule, or the surface protein CD70 and CD80. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises LIGHT. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD86.

[0009] In some embodiments, the engineered immune cell further comprises a third nucleic acid molecule encoding a molecule having transposase activity.

[0010] In some embodiments, the molecule is a transposase. In some embodiments, the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity. In some embodiments, the transposase is Sleeping Beauty transpose SB100X.

[0011] In some embodiments, the first nucleic acid molecule comprises a DNA. In some embodiments, the DNA is a DNA nanoplasmid comprising a transposon.

[0012] In some embodiments, the exogenous TCR molecule is stably expressed in the engineered immune cell. In some embodiments, the second nucleic acid molecule comprises an RNA. In some embodiments, the exogenous co-stimulatory molecule is transiently expressed in the immune cell.

[0013] In some embodiments, the third nucleic acid molecule comprises an RNA.

[0014] In some embodiments, the first nucleic acid molecule further encodes an enhancer. In some embodiments, the enhancer comprises a switch receptor. In some embodiments, the switch receptor is a PD1-41BB or a IL2 receptor. In some embodiments, the IL2 receptor comprises a mutation.

[0015] In some embodiments, the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a ligand for the exogenous co-stimulatory receptor molecule. In some embodiments, the ligand is CD27. In some embodiments, the ligand is CD28 or CTLA4. In some embodiments, the ligand is an exogenous ligand or an endogenous ligand.

[0016] In some embodiments, the first nucleic acid molecule or the second nucleic acid molecule is delivered into the engineered immune cell via electroporation or a particle. In some embodiments, the particle is a polyalkyleneimine or a lipid. In some embodiments, the particle is a lipid with a cationic headgroup. In some embodiments, the particle comprises a pH responsive lipid. In some embodiments, the particle comprises a PEGylated-lipid. In some embodiments, the particle is a lipid particular, polymer particle, or mixture thereof. In some embodiments, the particle is a nanoparticle. In some embodiments, the particle is a lipid nanoparticle (LPD), a lipoplex (LPX), a polyplex (PLX), or a lipopolyplex (LPLX) particle. In some embodiments, the particle further comprises at least one phosphatidylserine. In some embodiments, the particles are nanoparticles, in which: (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and / or (ii) the nanoparticles have a neutral or net negative charge and / or (iii) the zeta potential of the nanoparticles is 0 or less. In some embodiments, the particle comprises polyalkyleneimine. In some embodiments, (a) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) is 2.0 to 15.0; or (b) the molar ratio of the number of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) is at least about 48. In some embodiments, the ionic strength of the composition is about 50 mM or less. In some embodiments, the concentration of monovalent cationic ions is about 25 mM or less and the concentration of divalent cationic ions is about 20 pM or less. In some embodiments, the particle is a polyplex particle.

[0017] In some embodiments, the particle comprises a hydrophobic moiety having a binding moiety covalently attached thereto. In some embodiments, the hydrophobic moiety having a binding moiety covalently attached thereto and the particle are non-covalently associated with each other. In some embodiments, the hydrophobic moiety having a binding moiety covalently attached thereto is an integral part of the particle. In some embodiments, the hydrophobic moiety having a binding moiety covalently attached thereto comprises a polymer. In some embodiments, the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula I: L-X1-P-X2-B (I), wherein P comprises a polymer; L comprises a hydrophobic moiety attached to a first end of the polymer; Bcomprises a binding moiety attached to a second end of the polymer; XI is absent or a first linking moiety; and X2 is absent or a second linking moiety. In some embodiments, XI comprises a carbonyl group. In some embodiments, X2 comprises the reaction product of a maleimide group with a thiol or cysteine group of a compound comprising the binding moiety. In some embodiments, the hydrophobic moiety is or is comprised in a lipid. In some embodiments, the polymer provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion. In some embodiments, the polymer comprises polyethylene glycol (PEG). In some embodiments, the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula II:wherein B comprises the binding moiety.

[0018] In some embodiments, B comprises a moiety comprising the structure -N-peptide- C(O)-NH2.

[0019] In some embodiments, the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or an antibody derivative. In some embodiments, the particle is complexed with the nucleic acid molecules and / or encapsulates the nucleic acid molecules. In some embodiments, the engineered immune cell is administered to a subject in need thereof. In some embodiments, the subject has cancer.

[0020] In some embodiments, the engineered immune cell has been incubated with the exogenous peptide. In some embodiments, the engineered immune cell has been activated in the presence of the exogenous peptide in complex with the MHC molecule. In some embodiments, the exogenous peptide is a T cell epitope. In some embodiments, the T cell epitope is from a PRAME protein. In some embodiments, the T cell epitope comprises an amino acid sequence of SLLQHLIGL (SEQ ID NO: 116). In some embodiments, the MHC molecule is HLA 02:01. In some embodiments, the T cell epitope is from a RAS protein. In some embodiments, the RAS protein is a KRAS protein. In some embodiments, the KRAS protein comprises a mutation. In some embodiments, the mutation is a G12V mutation, a G12D mutation, or a G12C mutation.

[0021] In some embodiments, the T cell epitope comprises an amino acid sequence selected from the group consisting of VVGAVGVGK (SED ID NO: 51), VVVGAVGVGK (SED IDNO: 52), AVGVGKSAL (SED ID NO: 53), GADGVGKSAL (SED ID NO: 54), GAVGVGKSAL (SED ID NO: 55), GAVGVGKSA (SED ID NO: 56), and VVVGADGVGK (SED ID NO: 57).

[0022] IN some embodiments, the MHC molecule is HLA Al 1:01, HLA A03:01, HLA A68:01, HLA C01:02, HLA C03:03 / C03:04, HLA C05:01, or HLA Al 1:01.

[0023] In some embodiments, the peptide is not processed by the engineered immune cell.

[0024] In some embodiments, the first nucleic acid molecule comprises a promoter selected from the group consisting of EFl alpha, EFl alpha- Human T-lymphotropic virus 1 (HTLV), MP71, and MP71-HTLV. In some embodiments, the promoter is EFl alpha. In some embodiments, the promoter is EFlalpha-HTLV. In some embodiments, the promoter is MP71. In some embodiments, the promoter is MP71-HTLV. In some embodiments, the promoter is MP71-HTLV and co-electroporated with the first nucleic acid molecule.

[0025] In some embodiments, the first nucleic acid molecule is a TCR that recognizes an epitope from a PRAME protein in complex with an MHC molecule encoded by an HLA A02:01 allele.

[0026] In some embodiments, the TCR binds to a PRAME epitope in complex with an MHC encoded by an HLA A02:01 allele. In some embodiments, the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

[0027] In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105.

[0028] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104.

[0029] In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113.

[0030] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

[0031] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

[0032] In some embodiments, the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA Al 1:01 allele. In some embodiments, the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by an HLA Al 1:01 allele. In some embodiments, the TCR binds to the epitope comprising an amino acid sequence of VVGAVGVGK (SEQ ID NO: 51) in complex with an MHC encoded by an HLA Al 1:01 allele.

[0033] In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

[0034] In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

[0035] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identicalto SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

[0036] In some embodiments, the engineered immune cell further comprises a fourth nucleic acid molecule encoding the exogenous TCR for transiently expressing the exogenous TCR. In some embodiments, the fourth nucleic acid molecule comprises an RNA. In some embodiments, the exogenous TCR transiently expressed in the engineered immune cell increases cell expansion.

[0037] In some embodiments, the engineered immune cell is a population of engineered immune cells, and wherein, after being incubated for a period time, the number of the population of engineered immune cells is at least 2-fold, 3 -fold, 4-fold, 5 -fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more higher than the number of an otherwise identical population of engineered immune cells without the fourth nucleic acid molecule. In some embodiments, the population of engineered immune cells have been incubated for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days or more. In some embodiments, the population of engineered immune cells have been incubated for no more than 15 days, no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day or less.

[0038] In some embodiments, the population of engineered immune cells have been incubated in the presence of a cytokine. In some embodiments, the cytokine is IL-7 and / or IL-15.

[0039] In some embodiments, the engineered immune cell retains TCR expression and / or proliferative capacity with or without cryopreservation. In some embodiments, the engineered immune cell post cryopreservation exhibits comparable TCR expression and / or proliferative capacity compared to an otherwise identical engineered immune cell without cryopreservation.

[0040] In some embodiments, the engineered immune cell exhibits comparable or increased cytotoxicity towards tumor cells than an otherwise identical cell being retrovirally engineered.

[0041] In some embodiments, the engineered immune cell retains comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution. In some embodiments, the engineered immune cell retains comparable expansion capacity after being diluted by 1: 10 and 1:20.

[0042] In some embodiments, the engineered immune cell is administered into a subject in need thereof after being incubated ex vivo for no more than 15 days, no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day or less.

[0043] In some embodiments, the population of engineered immune cells have been incubated in the presence of a cytokine. In some embodiments, the cytokine is IL-7 and / or IL-15.

[0044] Provided herein is a cell culture comprising a population of immune cells comprising a plurality of engineered immune cells comprising a first engineered immune cell and a second engineered immune cell, wherein the first engineered immune cell of the plurality comprises a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR, and wherein the first and / or the second engineered immune cell of the plurality comprises a second nucleic acid molecule encoding an exogenous co-stimulatory molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed; and a peptide, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells for stimulating the first engineered immune cell.

[0045] In some embodiments, the peptide is presented on a surface protein of an engineered immune cell and / or a non-engineered immune cell of the population of immune cells. In some embodiments, the peptide is presented on a surface protein of a non-engineered immune cell of the population of immune cells. In some embodiments, the peptide is presented on a surface protein of an engineered immune cell of the population of immune cells. In some embodiments, the peptide is presented on a surface protein of the second engineered immunecell of the plurality. In some embodiments, the peptide is presented on an MHC molecule of an immune cell of the population of immune cells. In some embodiments, the peptide is a T cell epitope. In some embodiments, the exogenous co-stimulatory receptor molecule is transiently expressed.

[0046] In some embodiments, the exogenous co-stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC). In some embodiments, activation of the exogenous co-stimulatory receptor molecule provides a co-stimulatory signal to the engineered immune cell. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD70 and / or CD80. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD70 and CD80. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises LIGHT. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD86.

[0047] In some embodiments, the engineered immune cell further comprises a third nucleic acid molecule encoding a molecule having transposase activity. In some embodiments, the molecule is a transposase. In some embodiments, the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity. In some embodiments, the transposase is Sleeping Beauty transpose SB100X.

[0048] In some embodiments, the first nucleic acid molecule comprises a DNA. In some embodiments, the DNA is a DNA nanoplasmid comprising a transposon.

[0049] In some embodiments, the exogenous TCR is stably expressed in the immune cell. In some embodiments, the second nucleic acid molecule comprises an RNA. In some embodiments, the exogenous co-stimulatory molecule is transiently expressed in the immune cell. In some embodiments, the third nucleic acid molecule comprises an RNA.

[0050] In some embodiments, the first nucleic acid molecule further encodes an enhancer. In some embodiments, the enhancer comprises a switch receptor. In some embodiments, the switch receptor is a PD1-41BB or a IL2 receptor. In some embodiments, the IL2 receptor comprises a mutation.

[0051] In some embodiments, the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a ligand for the exogenous costimulatory receptor molecule. In some embodiments, the ligand is CD27. In some embodiments, the ligand is CD28 or CTLA4.

[0052] In some embodiments, the first nucleic acid molecule or the second nucleic acid molecule is delivered into the engineered immune cell via electroporation or a particle.

[0053] Provided herein is a method for producing an antigen specific T cell, the method comprising: (a) providing a population of immune cells comprising a plurality of engineered immune cells comprising a first engineered immune cell and a second engineered immune cell, wherein the first engineered immune cell of the plurality comprises a first nucleic acid encoding an exogenous TCR, and wherein the first and / or the second engineered immune cell of the plurality comprises a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed; and (b) culturing the population of immune cells in the presence of a peptide, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells, thereby stimulating the first engineered immune cell and producing the antigen specific T cell.

[0054] In some embodiments, the culturing is no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days or less. In some embodiments, the culturing is no more than 3 days. In some embodiments, the culturing is no more than 2 days.

[0055] Provided herein is a method for producing an antigen specific T cell, the method comprising: (a) providing a plurality of engineered immune cells; (b) culturing the plurality of engineered immune cells ex vivo to generate a therapeutically effective amount of antigen specific T cells, wherein the culturing is less than 7 days; and (c) administering the plurality of engineered immune cells into a subject in need thereof.

[0056] In some embodiments, the plurality of engineered immune cells comprises a first engineered immune cell and a second engineered immune cell, each engineered immune cell of the plurality comprises a first nucleic acid encoding an exogenous TCR, a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into agenomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous costimulatory receptor molecule is transiently expressed.

[0057] In some embodiments, culturing in (b) comprises culturing the plurality of engineered immune cells in the presence of a peptide. In some embodiments, the peptide is presented on the surface protein of the second engineered immune cell for stimulating the first engineered immune cell.

[0058] In some embodiments, the therapeutically effective amount of antigen specific T cells comprises at least 104T cells.

[0059] In some embodiments, providing the plurality of engineered immune cells comprises providing a population of immune cells from a subject; and delivering into the population of immune cells the first nucleic acid encoding the exogenous TCR, and the second nucleic acid encoding the exogenous co- stimulatory molecule, thereby generating the plurality of engineered immune cells.

[0060] In some embodiments, the culturing is less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days or less.

[0061] In some embodiments, the exogenous co- stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

[0062] In some embodiments, each engineered immune cell of the plurality further comprises a third nucleic acid encoding a transposase.

[0063] In some embodiments, further comprising, prior to delivering, selecting CD4+ T cells and / or CD8+ T cells from the population of immune cells.

[0064] In some embodiments, the population of immune cells are isolated from a sample from the subject. In some embodiments, the sample is a blood sample or a PBMC sample. In some embodiments, delivering comprises electroporating the first, the second, and / or the third nucleic acid molecule into the population of immune cells.

[0065] In some embodiments, delivering comprises using a particle to deliver the first, the second, and / or the third nucleic acid molecule into the population of immune cells. In some embodiments, the particle is a lipid nanoparticle (LNP), a lipoplex (LPX), a polyplex (PLX), a lipopolyplex (LPLX) particle, or any combination thereof. In some embodiments, the peptide is a T cell epitope. In some embodiments, the peptide is not further processed to be presented in complex with an MHC molecule.

[0066] In some embodiments, the antigen specific T cell comprises a plurality of antigen specific T cells. In some embodiments, the plurality of antigen specific T cells retain TCR expression and / or proliferative capacity with or without cry opreservation. In someembodiments, the plurality of antigen specific T cells post cryopreservation exhibits comparable TCR expression and / or proliferative capacity compared to an otherwise identical plurality of antigen specific T cells without cryopreservation.

[0067] In some embodiments, further comprising administering the plurality of antigen specific T cells into a subject in need thereof. In some embodiments, further comprising administering the plurality of antigen specific T cells into a subject in need thereof after no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days of culturing. In some embodiments, further comprising administering the plurality of antigen specific T cells into a subject in need thereof, and wherein the plurality of antigen specific T cells have been cryopreserved after no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days of culturing.

[0068] In some embodiments, the plurality of antigen specific T cells expand in the subject. In some embodiments, the plurality of antigen specific T cells expand better than retrovirally engineered T cells, and wherein the number of the plurality of antigen specific T cells after expansion is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10- fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or more, higher than the retrovirally engineered T cells after expansion when the same amount of cells are administered into the subject.

[0069] In some embodiments, the plurality of antigen specific T cells exhibit comparable or increased cytotoxicity towards tumor cells than retrovirally engineered T cells.

[0070] In some embodiments, the engineered immune cell retains comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution. In some embodiments, the engineered immune cell retains comparable expansion capacity after being diluted by 1: 10 and 1:20.

[0071] In some embodiments, the TCR binds to a PRAME epitope in complex with an MHC encoded by an HLA 02:01 allele. In some embodiments, the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

[0072] In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115.

[0073] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102.

[0074] In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

[0075] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

[0076] In some embodiments, the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA Al 1:01 allele. In some embodiments, the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by an HLA Al 1:01 allele. In some embodiments, the TCR binds to the epitope comprising an amino acid sequence of VVGAVGVGK (SEQ ID NO: 51) in complex with an MHC encoded by an HLA Al 1:01 allele.

[0077] In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6.

[0078] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

[0079] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 anda complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

[0080] In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

[0081] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

[0082] Provided herein is a method of treating a cancer in a subject in need thereof, the method comprising: (a) administering an ex vivo population of T cells comprising antigemMHC complex activated T cells, wherein the ex vivo population of T cells comprises at most 108T cells, wherein the antigemMHC complex activated T cells expand in vivo in the subject to at least 2-fold after administration to the subject or expand in vivo to at least 1010cells after administration to the subject.

[0083] Alternatively, or in addition, in some embodiments, further comprising, prior to (a), culturing the ex vivo population of T cells in the presence of an antigemMHC complex. In some embodiments, T cells are autologous T cells. In some embodiments, the ex vivo population of T cells are cultured ex vivo for no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days or less.

[0084] In some embodiments, the T cells comprises an engineered immune cell, wherein the engineered immune cell comprises a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; and a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co- stimulatory receptor molecule.

[0085] In some embodiments, (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous costimulatory receptor molecule is transiently expressed.

[0086] In some embodiments, the exogenous co- stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

[0087] In some embodiments, activation of the exogenous co- stimulatory receptor molecule provides a co-stimulatory signal to the engineered immune cell. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD70 and / or CD80. In some embodiments, the exogenous co- stimulatory receptor molecule or the surface protein comprises CD70 and CD80. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises LIGHT. In some embodiments, the exogenous co-stimulatory receptor molecule or the surface protein comprises CD86.

[0088] In some embodiments, the engineered immune cell further comprises a third nucleic acid molecule encoding a transposase. In some embodiments, the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity. In some embodiments, the transposase is Sleeping Beauty transpose SB100X.

[0089] In some embodiments, the first nucleic acid molecule comprises a DNA. In some embodiments, the DNA is a DNA nanoplasmid comprising a transposon. In some embodiments, the exogenous TCR is stably expressed in the immune cell. In some embodiments, the second nucleic acid molecule comprises an RNA. In some embodiments, the exogenous co-stimulatory molecule is transiently expressed in the immune cell. In some embodiments, the third nucleic acid molecule comprises an RNA.

[0090] In some embodiments, the first nucleic acid molecule further encodes an enhancer. In some embodiments, the enhancer comprises a switch receptor. In some embodiments, the switch receptor is a PD1-41BB or a IL2 receptor. In some embodiments, the IL2 receptor comprises a mutation.

[0091] In some embodiments, the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an exogenous ligand for the exogenousco-stimulatory receptor molecule. In some embodiments, the exogenous ligand is CD27. In some embodiments, the exogenous ligand is CD28 or CTLA4.

[0092] In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen comprises a mutation. In some embodiments, the T cells are produced by the method of producing an antigen specific T cell.

[0093] Provided herein is a method for producing a population of immune cells comprising a plurality of antigen specific T cells, the method comprising: (a) providing a population of at least IxlO9immune cells; (b) introducing into immune cells of the population of immune cells a first nucleic acid encoding an exogenous TCR and a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, thereby producing a population of immune cells comprising a plurality of engineered T cells, wherein the plurality of engineered T cells comprises a first engineered immune cell and a second engineered immune cell, wherein (i) the first engineered immune cell comprises the first nucleic acid, (ii) the first and / or the second engineered immune cell comprises the second nucleic acid, and (iii) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or (iv) the exogenous co-stimulatory receptor molecule is transiently expressed; (c) expanding the population of immune cells comprising the plurality of engineered T cells in the presence of a peptide for a first time period of 2 days, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells comprising the plurality of engineered T cells and a complex comprising the surface protein and the peptide is recognized by the an exogenous TCR, thereby expanding the first engineered immune cell and producing the population of immune cells comprising a plurality of antigen specific T cells, wherein (d) the percentage of T cells comprising the first nucleic acid is at least 10% of the total number of T cells in the population of immune cells at the first time period, and / or (e) when the population of immune cells comprising the plurality of engineered T cells is expanded in the presence of the peptide for a second time period of 5 or more days the percentage of T cells comprising the first nucleic acid is at least 50% of the total number of T cells in the population of immune cells at the second time period.

[0094] In some embodiments, providing in (a) comprises providing a population of at least 2xl09immune cells.

[0095] In some embodiments, after (b) and before (c), the population of immune cells comprising a plurality of engineered T cells comprises less than 20% viable immune cells.

[0096] In some embodiments, expanding in (c) comprises preferentially expanding the first engineered immune cell. In some embodiments, the immune cells without the first nucleic acid are not substantially expanded in step (c).

[0097] Provided herein is an ex vivo activated and / or expanded population of T cells produced by the method described herein. In some embodiments, the ex vivo activated and / or expanded population of T cells are cultured for no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days.

[0098] Provided herein is a pharmaceutical composition comprising the engineered immune cell described herein, and a pharmaceutically acceptable carrier.

[0099] Provided herein is the use of a plurality of T cells for treating a cancer in a subject in need thereof, comprising administering at most 106T cells, wherein the T cells have been activated ex vivo in the present of an antigen in complex with an MHC molecule, wherein the T cells expand in vivo in the subject to at least 2-fold or at least 1010cells.

[0100] Provided herein is the use of the ex vivo activated and / or expanded population of T cells, the engineered immune cell, or the engineered immune cell in the manufacture of a medicament for treating cancer.

[0101] In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia, n some embodiments, the cancer is selected from the group consisting of bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, carcinoma of the sexual and reproductive organs, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors glioma, meningioma, and pituitary adenoma. In some embodiments, the cancer is a solid cancer.INCORPORATION BY REFERENCE

[0102] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated byreference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0104] FIG. 1 depicts the Insta TCR process described in the present disclosure. Patient apheresis derived T cells can be enriched via CD4 / CD8 microbeads and then electroporated with TCR Nanoplasmid, mRNAs coding for Sleeping Beauty (SB IOOx) transposase, and costimulatory molecules CD70 and CD80. Cells can be kept in culture for 2 days in vitro to provide effective antigen specific T cell activation.

[0105] FIG. 2A depicts experimental data of PRAME TCR expression measured by flow cytometry. FIG. 2B shows the T cell counts on Day 10 of healthy donor cells electroporated with indicated components. Insta TCR cells were kept in extended culture beyond Day 2 with supportive cytokines IL-7 and IL- 15. Conditions highlighted in the black boxes are the Insta TCR process.

[0106] FIG. 3A depicts experimental data of PRAME TCR expression measured by flow cytometry on Day 6, 10, and 13 post electroporation. No freeze thaw (No FT) conditions are depicted in the top panel and freeze thaw (FT) conditions are depicted in the bottom panel. FIG. 3B shows T cell growth monitored post electroporation for No FT and FT conditions.

[0107] FIG. 4 depicts representative flow cytometry measuring PRAME TCR expression monitored on Day 2, 7, and 14 post-electroporation. The Insta TCR T cells shown are mixed with CTV labelled autologous PBMCs at ratio 1: 10, 1:20, or left undiluted.

[0108] FIGs. 5A-5B show cytotoxicity of Insta TCR T cells and retrovirally engineered TCR T cells by monitoring 3D tumor cell spheroids using Incucyte device. FIG. 5A depicts results in the SKMEL5 cell line. FIG. 5B depicts results in a U2OS cell line. FIG. 5C depicts both SKMEL5 and U2OS cells expressing PRAME TCR on day 5 using a flow cytometry plot. Cell counts were compared between untransduced cells as the control, retroviral engineered TCR T cells, and Insta TCR T cells.

[0109] FIG. 6A depicts 624Mel tumor volume in a mouse following treatment with T cells manufactured by different manufacturing processes with different promoters. RV TCR-MP71 [3e6] represents retrovirally engineered TCR-MP71 at a concentration of 3,000,000 cells, RVTCR-MP71 [le6] represents retrovirally engineered TCR-MP71 at a concentration of 1,000,000 cells, InstaTCR 2 day-EFal [3e6] represents Insta TCR EFal cells at a concentration of 3,000,000 cells in culture for 2 days, and InstaTCR 10 day-EFal [3e6] represents Insta TCR EFal cells at a concentration of 3,000,000 cells in culture for 10 days. An untransduced treatment group was used as a negative control. Tumor cells were subcutaneously injected into NCG mice. After the tumor grew to approximately 100mm3, T cells were injected intravenously into mice and tumor growth was monitored. FIG. 6B depicts 624Mel tumor volume in a mouse following treatment with RV TCR-MP71 [3e6]. FIG. 6C depicts 624Mel tumor volume in a mouse following treatment with Insta TCR 10 day-EFal [3e6] in culture for 2 days. FIG. 6D depicts 624Mel tumor volume in a mouse following treatment Insta TCR 2 day-EFal [3e6] in prolonged culture for 10 days.

[0110] FIG. 7A depicts experimental data of PRAME TCR expression measured by flow cytometry on Day 2, 8, 10, and 14 days post electroporation with either EFla-HTLV TCR or MP71 TCR. FIG. 7B shows T cell growth post electroporation for EFla-HTLV TCR or MP71 TCR conditions.

[0111] FIGs. 8A-8B depict the Insta TCR process carried out with three different promoters. FIG. 8A shows T cell expansion of promoters EFla-HTLV, MP71 TCR, and MP71-HTLV on Day 2, 5, 8, and 14. FIG. 8B shows PRAME TCR expression of promoters EFla-HTLV, MP71 TCR, and MP71-HTLV on Day 2,5, 8, and 14.

[0112] FIG. 9 depicts T cell expansion after PRAME A02 TCR mRNA was coelectroporated in MP71 promoter Insta TCR process. The total live cells were monitored on Day 2, 5, 8, and 14.

[0113] FIG. 10A depicts the effectiveness of costimulatory molecules CD80+CD70, CD80, CD70, LIGHT, and CD86 measured by fold expansion. FIG. 10B depicts the total TCR expressing T cells under the various costimulatory molecules CD80+CD70, CD80, CD70, LIGHT, and CD86 days 2, 5, 9, and 13 after electrorotation.

[0114] FIG. 11 depicts experimental data of TCR T cells specifically enriched for HLA A* 11 :01 / G12V KRAS TCR. TCR T cells have been prepared by three different processes including electroporation with TCR nanoplasmid and Sleeping Beauty (SB IOOx) transposase mRNA, Pan T Cell activation, and the Insta TCR process.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0115] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0116] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the pertinent art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0117] The term “Insta TCR” or “InstaTCR” as used herein, refers to the T cell manufacturing process or processes described in the present disclosure.

[0118] The term “Insta TCR T cell” or “InstaTCR T cell” as used herein, refers to the T cell produced by the T cell manufacturing processes described in the present disclosure.

[0119] An antigen is a foreign substance to the body that induces an immune response. A “neoantigen” refers to a class of tumor antigens which arise from tumor- specific changes in proteins. Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a substitution in a protein sequence, a frame shift mutation, a fusion polypeptide, an in-frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.

[0120] A “neoepitope” refers to an epitope that is not present in a reference, such as a nondiseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where a corresponding epitope is found in a normal nondiseased cell or a germline cell but, due to one or more mutations in a diseased cell, e.g., a cancer cell, the sequence of the epitope is changed so as to result in the neoepitope.

[0121] A “mutation” refers to a change of or a difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition or deletion) compared to a reference nucleic acid. A “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. In some embodiments, a mutation is a non-synonymous mutation. A “non- synonymous mutation” refers to a mutation, for (e.g., a nucleotide substitution), which does result in an amino acid change such as an amino acid substitution in the translation product. A “frameshift” occurs when a mutation disrupts the normal phase of a gene’s codon periodicity (also known as “reading frame”), resulting in translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.

[0122] “Antigen processing” or “processing” refers to the degradation of a polypeptide or antigen into procession products, which are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells.

[0123] An “antigen presenting cell” (APC) refers to a cell which presents peptide fragments of protein antigens in association with MHC molecules on its cell surface. The term includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes). In some cases, the APC can be a cancer cell.

[0124] The term “affinity” refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). KD refers to the dissociation constant between two members of a binding pair and has units of molarity. KA refers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore™ SPR units. Koff refers to the off-rate constant of two members of a binding pair, (e.g., the off-rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR). Konrefers to the on- rate constant of two members of a binding pair, (e.g., the on-rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR).

[0125] Throughout this disclosure, “binding data” results may be expressed in terms of an “IC50.” Affinity may also be expressed as the inhibitory concentration 50 (IC50), or the concentration at which 50% of a first member of a binding pair (e.g., a peptide) is displaced. Likewise, ln(IC5o) refers to the natural log of the IC50. For example, an IC50 may be the concentration of a tested peptide in a binding assay at which 50% inhibition of binding of a labeled reference peptide is observed. Given the conditions in which the assays are run (e.g., limiting HLA protein concentrations and / or labeled reference peptide concentrations), these values can approximate KD values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. Binding can also be determined using other assay systems including those using live cells (e.g.,Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147: 189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall etal., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268: 15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149: 1896 (1992)).

[0126] The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or nonnatural amino acid residues such as cyclohexylalanine. A derived or prepared epitope can be an analog of a native epitope. The term “derived from” refers to the origin or source, and may include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells. For example, an expanded or induced antigen specific T cell may be derived from a T cell. For example, an expanded or induced antigen specific T cell may be derived from an antigen specific T cell in a biological sample. For example, a matured APC (e.g., a professional APC) may be derived from a non-matured APC (e.g., an immature APC). For example, an APC may be derived from a monocyte (e.g., a CD14+monocyte). For example, a dendritic cell may be derived from a monocyte (e.g., a CD14+monocyte). For example, an APC may be derived from a bone marrow cell.

[0127] An “epitope” is the collective features of a molecule (e.g., a peptide’s charge and primary, secondary and tertiary structure) that together form a site recognized by another molecule (e.g., an immunoglobulin, T cell receptor, HLA molecule, or chimeric antigen receptor). For example, an epitope can be a set of amino acid residues involved in recognition by a particular immunoglobulin; a Major Histocompatibility Complex (MHC) receptor; or in the context of T cells, those residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from a natural source, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, amino acid mimetics, (such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues). Throughout this disclosure,epitopes may be referred to in some cases as peptides or peptide epitopes. In certain embodiments, there is a limitation on the length of a peptide of the present disclosure. The embodiment that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises a region (z.e., a contiguous series of amino acid residues) having 100% sequence identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% sequence identity with a native peptide sequence. Thus, for a peptide comprising an epitope described herein and a region with 100% sequence identity with a native peptide sequence, the region with 100% sequence identity to a native sequence generally has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an “epitope” described herein is comprised by a peptide having a region with less than 51 amino acid residues that has 100% sequence identity to a native peptide sequence, in any increment down to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0128] A “T cell epitope” refers to a peptide sequence bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. A peptide-MHC complex can be recognized and bound by a TCR of a T cell (e.g., a cytotoxic T-lymphocyte or a T-helper cell).

[0129] A “T cell” includes CD4+T cells and CD8+T cells. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells. T cells may be generated by the method described in the application, for a clinical application. T cells or adoptive T cells referred to here, such as for a clinical application are cells isolated from a biological source, manipulated and cultured ex vivo and prepared into a drug candidate for a specific therapy such as a cancer, e.g., melanoma. When drug candidate cells pass specific qualitative and quantitative criteria for fitness for a clinical application, the drug candidate may be designated a drug product. In some cases, a drug product is selected from a number of drug candidates. In the context of this application, a drug product is a T cell, more specifically, a population of T cells, or more specifically a population of T cells with heterogeneous characteristics and subtypes. For example, a drug product, as disclosed herein may have a population of T cells comprisingCD8+ T cells, CD4+ T cells, with cells at least above a certain exhibiting antigen specificity, a certain percentage of each exhibiting a memory phenotype, among others.

[0130] An “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0131] An “immunogenic” peptide or an “immunogenic” epitope or an “immunogenic” peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, for example, a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response and / or a B lymphocyte response. Immunogenic peptides described herein are capable of binding to an HLA molecule and thereafter induce a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response, or a HTL response) to the peptide.

[0132] A “protective immune response” or “therapeutic immune response” refers to a CTL and / or an HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0133] A “T cell receptor” (“TCR”) refers to a molecule, whether natural or partly or wholly synthetically produced, found on the surface of T lymphocytes (T cells) that recognizes an antigen bound to a major histocompatibility complex (MHC) molecule. The ability of a T cells to recognize an antigen associated with various diseases (e.g., cancers) or infectious organisms is conferred by its TCR, which is made up of both an alpha (a) chain and a beta (P) chain or a gamma (y) and a delta (5) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendous diversity of the TCR. This multi-subunit immune recognition receptor associates with the CD3 complex and binds peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of a TCR to a peptide on an APC is a central event in T cell activation.

[0134] “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. The terms “major histocompatibility complex” and the abbreviation “MHC” can include any class of MHC molecule, such as MHC class I and MHC class II molecules, and relate to a complex of genes which occurs in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, a “Human Leukocyte Antigen” or “HLA” refers to a human Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, el al.,Immunology, 8THEd., Lange Publishing, Los Altos, Calif. (1994). For a detailed description of the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3rdEd., Raven Press, New York (1993).

[0135] The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or foreign antigens and thus for regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors. The proteins encoded by the MHC can be expressed on the surface of cells and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T-cell. MHC binding peptides can result from the proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes, (e.g., T cell epitope and B cell epitope). MHCs can transport the peptides to the cell surface and present them there to specific cells, such as cytotoxic T-lymphocytes, T-helper cells, or B cells. The MHC region can be divided into three subgroups, class I, class II, and class III. MHC class I proteins can contain an a-chain and P2- microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T-cells. MHC class II proteins can contain a- and P-chains and they can present antigen fragments to T-helper cells. MHC class III region can encode for other immune components, such as complement components and cytokines. The MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0136] A “receptor” refers to a biological molecule or a molecule grouping capable of binding a ligand. A receptor may serve, to transmit information in a cell, a cell formation or an organism. A receptor comprises at least one receptor unit, for example, where each receptor unit may consist of a protein molecule. A receptor has a structure which complements that of a ligand and may complex the ligand as a binding partner. The information is transmitted in particular by conformational changes of the receptor following complexation of the ligand on the surface of a cell. In some embodiments, a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of suitable length. A “ligand” refers to a molecule which has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In some embodiments, a ligand is to be understood as meaning a peptide or peptide fragment which has a suitable length and suitable binding motifs in its aminoacid sequence, so that the peptide or peptide fragment is capable of forming a complex with MHC proteins such as MHC class I or MHC class II proteins. In some embodiments, a “receptor / ligand complex” is also to be understood as meaning a “receptor / peptide complex” or “receptor / peptide fragment complex”, including a peptide- or peptide fragment-presenting MHC molecule such as MHC class I or MHC class II molecules.

[0137] A “native” or a “wild type” sequence refers to a sequence found in nature. The term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0138] The terms “peptide” and “peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acid residues. A “synthetic peptide” refers to a peptide that is obtained from a nonnatural source, e.g., is man-made. Such peptides can be produced using such methods as chemical synthesis or recombinant DNA technology. “Synthetic peptides” include “fusion proteins.”

[0139] The term “motif’ refers to a pattern of residues in an amino acid sequence of defined length, for example, a peptide of less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, from about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchor residues. In some embodiments, an MHC class I motif identifies a peptide of 7, 8 9, 10, 11, 12 or 13 amino acid residues in length. In some embodiments, an MHC class II motif identifies a peptide of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acid residues in length. A “cross-reactive binding” peptide refers to a peptide that binds to more than one member of a class of a binding pair members (e.g., a peptide bound by both a class I HLA molecule and a class II HLA molecule).

[0140] The term “residue” refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or that is encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows the conventional practice. The amino group is presented to the left (the amino-or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope, they are numbered in an amino to carboxyl direction with the first position being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L- form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acid residues having D-forms is represented by a lower-case single letter or a lower case three letter symbol. However, when three letter symbols or full names are used without capitals, they can refer to L amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or “G”. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.)

[0141] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate peptide function are well-known in the art.

[0142] “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and / or physiologically compatible composition or component of a composition. A “pharmaceutical excipient” or “excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.

[0143] According to the present disclosure, the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, for example, a cellular or humoral immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease. The term “individualized cancer vaccine” or “personalized cancer vaccine” “personal cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0144] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA, for example, mRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments, the polynucleotide that is administered using the methods of the invention is mRNA.

[0145] The terms “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their in situ environment. For example, an “isolated” epitope can be an epitope that does not include the whole sequence of the protein from which the epitope was derived. For example, a naturally-occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector, and / or such a polynucleotide or peptide could be part of a composition, and still be “isolated” in that such vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. The term “substantially pure” as used herein refers to material which is at least 50% pure (z.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0146] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when comparedand aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as an amino acid sequence of a peptide or a coding region of a nucleotide sequence.

[0147] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0148] The terms “effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a therapeutic effective to “treat” a disease or disorder in a subject or mammal. The therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0149] The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. The “stimulation”refers to a response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event. For example, stimulation of a T cell can refer to binding of a TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell can refer to a step within protocol 1 or protocol 2 in which PBMCs are cultured together with peptide loaded APCs.

[0150] The term “enriched” refers to a composition or fraction wherein an object species has been partially purified such that the concentration of the object species is substantially higher than the naturally occurring level of the species in a finished product without enrichment. The term “induced cell” refers to a cell that has been treated with an inducing compound, cell, or population of cells that affects the cell’s protein expression, gene expression, differentiation status, shape, morphology, viability, and the like.

[0151] A “reference” can be used to correlate and / or compare the results obtained in the methods of the present disclosure from a diseased specimen. Typically, a “reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a disease, either obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A “reference” can be determined empirically by testing a sufficiently large number of normal specimens.

[0152] As used herein, a tumor unless otherwise mentioned, is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document. While a tumor is a cancer of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, leukemia.Overview

[0153] Conventional engineered cell therapy manufacturing comprises polyclonal T cell activation followed by transduction with one or more transgenes, typically with retrovirus or lentivirus. While this approach can lead to efficiency of engineering and large numbers of engineered cells, it usually needs extensive time in cell culture (for example, 1-3 weeks or more), which can be both expensive and can negatively affect the fitness of the engineered cells. By contrast, short manufacturing protocols (for example, less than 3 days) can maintain the fitness of cells but may lead to low engineering efficiency.

[0154] To overcome these challenges, the present disclosure provides novel transposon-based platforms in which transgenes can be introduced into unactivated cells and then stimulated to expand in an antigen- specific manner. In the methods provided herein (termed “Insta TCR”) T cells from a leukapheresis can be electroporated with the Sleeping Beauty lOOx (SB100X) transposase encoded by mRNA, and a DNA nanoplasmid carrying a transposon that encodesat least the TCR construct. The T cells can then be activated with the antigens in complex with MHC molecules. In this way, cells that express the TCR can encounter their antigen and can be preferentially stimulated to survive and expand, enriching engineered cells.

[0155] Since the TCR can recognize the epitope presented on HLA, the cognate peptide to be presented on the natural HLA can be added to the cell culture comprising the engineered T cells. The mRNA may be delivered to the engineered T cells for expressing the antigens, but the antigens expressed may need to be actively processed and presented by the engineered T cells, which can be inefficient in T cells. Additionally, because the TCR construct may not have co-stimulatory molecules as part of the TCR construct as is the case with the CAR construct (e.g., 4- IBB intracellular signaling domain downstream of the CAR), co-stimulatory molecules such as CD70 and / or CD80 can be delivered into the engineered T cells via mRNA to facilitate activation of the engineered T cells. Peptides comprising T cell epitopes can be supplied in the cell culture and be presented by the HLA on the surface the engineered T cells. In this way, the engineered T cells in the culture can function as efficient antigen-presenting cells. To enable the efficiency of this process, many parameters can be optimized, including but not limited to electroporation approach, plasmid design, promoter selection, cell handling parameters, peptide concentration, mRNA concentration, cell selection method, amongst others.

[0156] The InstaTCR manufacturing process may take up to three days while the overall time in culture for the T cells may be as short as 2 days (approximately 48 hours). The short culture period may enable the T cells to maintain a desirable fitness state. The short time in culture may enable the cells to expand more rapidly upon infusion at a higher rate than those cells engineered with a longer process. The short ex vivo process may also enable higher throughput and faster treatment of critically ill patients by decreasing the overall turnaround time for the product. The InstaTCR process can allow for larger transgene cargo than traditional viral approaches and insertion into the genome of T cells without strong pre-activation. The transposon encoding the T cell receptor and molecular enhancers may be introduced into the cells prior to activation, which may allow for stimulation to occur directly to the engineered T cell receptor rather than broad T-cell activation.Engineered Immune Cells

[0157] The present disclosure provides engineered immune cells. The engineered immune cells can be manufactured by the Insta TCR methods described herein.

[0158] Described herein are engineered immune cells comprising TCRs. In some embodiments, disclosed herein are engineered immune cells comprising a nucleic acid molecule encoding a T-cell receptor (TCR). In some embodiments, the TCR can bind to KRAS, PRAME, or GAT A3.

[0159] The engineered immune cell provided herein can comprise a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule. The engineered immune cell can comprise a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule. The second nucleotide sequence may not be integrated into a genomic nucleic acid molecule of the engineered immune cell. In some cases, the exogenous co-stimulatory receptor molecule can be transiently expressed. The exogenous co-stimulatory receptor molecule can function to facilitate activation of the engineered immune cell. For example, the exogenous co-stimulatory receptor molecule can comprise one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. The engineered immune cell can further comprise an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell. The exogenous TCR molecule can bind to the exogenous peptide when presented in complex with the MHC.

[0160] The engineered immune cell provided herein can comprise a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule. The engineered immune cell provided herein can comprise a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule. The engineered immune cell can comprise an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell. The exogenous TCR molecule may bind to the exogenous peptide when presented in complex with the MHC.

[0161] The engineered immune cell provided herein can further comprise the second nucleotide sequence. The second nucleotide sequence may not be integrated into a genomic nucleic acid molecule of the engineered immune cell. In some cases, the exogenous co- stimulatory receptor molecule may be transiently expressed. The engineered immune cell can further comprise an exogenous co-stimulatory receptor molecule. The exogenous co- stimulatory receptor molecule can be a surface protein endogenously expressed on an antigen presenting cell (APC). The engineered immune cell can lead to the activation of the exogenous co-stimulatory receptor molecule. The exogenous co-stimulatory receptor molecule can provide a co-stimulatory signal to the engineered immune cell. In some cases, the engineered immune cell can comprise an exogenous co-stimulatory receptor molecule or the surfaceprotein. The exogenous co-stimulatory receptor molecule or the surface protein can further comprise one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. In some cases, the exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and / or CD80. In some cases, the exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and CD80. In some cases, the exogenous co-stimulatory receptor molecule or the surface protein can comprise LIGHT. In some cases, the exogenous co-stimulatory receptor molecule or the surface protein can comprise CD86.

[0162] The engineered immune cell provided herein can further comprise a third nucleic acid molecule encoding a molecule having transposase activity. The engineered immune cell can further comprise a molecule that may be a transposase. The transposase can comprise Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity. In some cases, the transposase can comprise Sleeping Beauty transpose SB100X.

[0163] The engineered immune cell provided herein can further comprise the first nucleic acid molecule. The first nucleic acid molecule may comprise a DNA. The DNA can comprise a DNA nanoplasmid comprising a transposon.

[0164] The engineered immune cell can further comprise an exogenous TCR molecule. The TCR molecule may be stably expressed in the engineered immune cell.

[0165] The engineered immune cell can comprise the second nucleic acid molecule. The second nucleic acid molecule can comprise an RNA. The engineered immune can further comprise an exogenous co-stimulatory molecule. In some cases, the engineered immune cell comprising the exogenous co-stimulatory receptor can be transiently expressed. The engineered immune cell can further comprise the third nucleic acid molecule. The third nucleic acid molecule can comprise an RNA.

[0166] The first nucleic acid molecule can further encode an enhancer. The enhancer can comprise a switch receptor. The switch receptor can comprise a PD1-41BB or an interleukin- 2 receptor (or IL2 receptor or IL2R). In some cases, the IL2 receptor can comprise a mutation (e.g., a IL2R variant as provided herein). The present disclosure also provides variants of the interleukin-2 (IL2). In one embodiment, the IL2 variants described herein have amino acid substitutions at the region of IL2 that contacts the alpha (a) subunit of the heterotrimeric IL2 receptor complex, IL2Rabg, reducing its ability to bind and activate the heterotrimeric receptor complex. Conversely, the corresponding IL2Ra variants described herein haveamino acid substitutions compensating for such reduced ability of IL2 variants to bind to and activate IL2Rabg, preferably at amino acid residues contacted by IL2 amino acid residues that are substituted in the IL2 variants described herein. The IL2 variants show impaired binding to and / or activation of wild type IL2R,IL2R comprising the (wild type) alpha subunit of IL2R. However, variation in the alpha subunit of IL2R at least partially restores binding to and / or activation of IL2R comprising a variant of the alpha subunit of IL2R. Thus, described herein are pairs, sets or systems of corresponding variants of alpha subunits of IL2R and IL2 which show a level of binding and / or activation which exceeds the level of binding and / or activation shown by the variants of IL2 and wild type IL2Rabg.

[0167] In different embodiments, the human alpha subunit of IL2R or the functional variant thereof and the human IL2 or the functional variant thereof are substituted at least the following positions (relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and relative to wild type human IL2 and numbered in accordance with wild type human IL2): (i) IL2R or functional variant thereof: position 1 (glutamic acid), and IL2 or functional variant thereof: position 35 (lysine); (ii) IL2R or functional variant thereof: position 29 (glutamic acid), and IL2 or functional variant thereof: position 43 (lysine) ;(iii) IL2R or functional variant thereof: position 38 (lysine), and IL2 or functional variant thereof: position 61 (glutamic acid); (iv) IL2R or functional variant thereof: position 1 (glutamic acid), IL2 or functional variant thereof: position 35 (lysine), IL2R or functional variant thereof: position 29 (glutamic acid), and IL2 or functional variant thereof: position 43 (Lysine); (v) IL2R or functional variant thereof: position 1 (glutamic acid), IL2 or functional variant thereof: position 35 (Lysine), IL2R or functional variant thereof: position 38 (Lysine), and IL2 or functional variant thereof: position 61 (glutamic acid); (vi) IL2R or functional variant thereof: position 29 (glutamic acid), IL2 or functional variant thereof: position 43 (Lysine), IL2R or functional variant thereof: position 38 (Lysine), and IL2 or functional variant thereof: position 61 (glutamic acid); or (vii) IL2R or functional variant thereof: position 1 (glutamic acid), IL2 or functional variant thereof: position 35 (lysine), IL2R or functional variant thereof: position 29 (glutamic acid), IL2 or functional variant thereof: position 43 (Lysine), IL2R or functional variant thereof: position 38 (Lysine), and IL2 or functional variant thereof: position 61 (glutamic acid).

[0168] The nucleic acid molecule can comprise a nucleotide sequence encoding a ligand for the exogenous co-stimulatory receptor molecule. In some cases, the ligand may be CD27. In some cases, the ligand may be CD28 or CTLA4. In some cases, the ligand may be an exogenousligand or an endogenous ligand. In some cases, the ligand may be an endogenous ligand (e.g., endogenously expressed by the engineered immune cell).

[0169] The engineered immune cell can further comprise the first nucleic acid molecule or the second nucleic acid molecule. The first nucleic acid molecule or second nucleic acid molecule may be delivered into the engineered immune cell via electroporation or a particle.

[0170] In some cases, delivery may take place using biological, chemical, mechanical, or physical methods. In some cases, biological methods may comprise viral vectors. In some cases, chemical methods may comprise calcium phosphate coprecipitation. In some cases, mechanical methods may comprise microinjection. In some cases, physical methods may comprise electroporation, magnetoporation, sonoporation, or optoporation. In some cases, physical methods may comprise of electroporation.

[0171] The engineered immune cell can comprise a particle. A particle can comprise a polyalkyleneimine or a lipid. The particle can comprise a lipid with a cationic headgroup. The particle can comprise a pH responsive lipid. The particle can comprise a PEGylated-lipid. The particle can comprise a lipid particular, polymer particle, or mixture thereof.

[0172] The particle can comprise a nanoparticle. The particle can comprise a lipid nanoparticle (LPD), a lipoplex (LPX), a polyplex (PLX), or a lipopolyplex (LPLX) particle. The particle can further comprise at least one phosphatidylserine.

[0173] The particles can further comprise nanoparticles. In some cases, the number of positive charges in the nanoparticles may not exceed the number of negative charges in the nanoparticles. In some cases, the nanoparticles may have a neutral or net negative charge. In some cases, the zeta potential of the nanoparticles may be 0 or less.

[0174] The particle may comprise a polyalkyleneimine comprising the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) may be 2.0 to 15.0. In some cases, the molar ratio of the number of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) may be at least about 48. In some cases, the ionic strength of the composition may be 50 mM or less. In some cases, the concentration of monovalent cationic ions may be 25 mM or less. In some cases, the concentration of divalent cationic ions may be 20 pM or less.

[0175] The particle can comprise a polyplex particle. The particle can comprise a hydrophobic moiety having a binding moiety covalently attached thereto. In some cases, the particle can benon-covalently associated with each other. In some cases, the binding moiety covalently attached thereto can be an integral part of the particle.

[0176] The hydrophobic moiety having a binding moiety covalently attached thereto can comprise a polymer. The hydrophobic moiety having a binding moiety covalently attached thereto can comprise a compound of Formula L-X1-P-X2-B (I). In some cases, P can comprise a polymer. In some cases, L can comprise a hydrophobic moiety attached to a first end of the polymer. In some cases, B can comprise a binding moiety attached to a second end of the polymer. In some cases, XI may be absent or a first linking moiety. In some cases, X2 may be absent or a second linking moiety. The XI can comprise a carbonyl group. The X2 can comprise the reaction product of a maleimide group with a thiol or cysteine group of a compound. In some cases, X2 can further comprise the binding moiety.

[0177] The hydrophobic moiety can be or can be comprised in a lipid. The engineered immune cell can comprise a polymer that provides stealth property, extends circulation half-life, and / or reduces non-specific protein binding or cell adhesion. The polymer can comprise polyethylene glycol (PEG). In some cases, the polymer comprising polyethylene glycol (PEG) can comprise a hydrophobic moiety having a binding moiety covalently attached thereto comprising a compound of Formula II.

[0178] In some cases, B can comprise the binding moiety. In some cases, B can comprise a moiety comprising the structure -N-peptide-C(O)-NH2. In some cases, the binding moiety covalently attached to the hydrophobic moiety can comprise an antibody or an antibody derivative.

[0179] The particle can be complexed with the nucleic acid molecules and / or encapsulates the nucleic acid molecules.

[0180] The engineered immune cell can be administered to a subject in need thereof. In some cases, the subject may have cancer.Engineered Immune Cells Comprising T Cell Receptors (TCRs)

[0181] Described herein are engineered immune cells comprising TCRs. In some embodiments, disclosed herein are engineered immune cells comprising a nucleic acidmolecule encoding a T-cell receptor (TCR). In some embodiments, the TCR can bind to an epitope from RAS (e.g., KRAS) or PRAME in complex with an MHC molecule.

[0182] In some embodiments, the engineered immune cell TCR can bind to a PRAME epitope in complex with an MHC encoded by the HLA-A:02:01. In some embodiments, the engineered immune cell TCR can bind to a RAS epitope in complex with an MHC.

[0183] In some embodiments, the TCR can comprise TCR sequences described in Table 1 and Table 2. The signal peptides in the TCR chains of Table 1 are bolded. The TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, 70%, 80%, 90%, 95%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences in Table 1, and / or a TCR beta variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences in Table 1. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences in Table 1, and / or a TCR beta chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences in Table 1.

[0184] The TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences (without the signal peptides) in Table 1, and / or a TCR beta variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domainsequences (without the signal peptides) in Table 1. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences (without the signal peptides) in Table 1, and / or a TCR beta chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences (without the signal peptides) in Table 1.

[0185] The TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences in Table 2, and / or a TCR beta variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences in Table 2. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences in Table 2, and / or a TCR beta chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences in Table 2.

[0186] The TCR described herein can comprise a TCR alpha variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha variable domain sequences (without the signal peptides) in Table 2, and / or a TCR beta variable domain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta variable domain sequences (without the signal peptides) in Table 2. The soluble TCR described herein can comprise a TCR alpha chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR alpha chain sequences (without the signal peptides) in Table 2, and / or a TCR beta chain having a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of the TCR beta chain sequences (without the signal peptides) in Table 2.

[0187] In some embodiments, the TCR can comprise a beta chain construct and an alpha chain construct. In some embodiments, the TCR beta chain construct of the engineered immune cell can comprise a complementarity determining region 3 (CDR3). In some embodiments, the CDR3 can have an amino acid sequence CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the recombinant nucleic acid encodes a TCR that recognizes an HLA-A02:01 epitope (e.g., an epitope having an amino acid sequence as set forth in SEQ ID NO: 116). In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequencewith at least 80% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 24, 40, 58-80. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 114. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 104.

[0188] In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:100. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein theCDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:101. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein theCDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99%, or 100% sequence identity to an amino acid sequence CAGLADYGGSQGNLIF (SEQ ID NO: 102).

[0189] In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 106. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 112.

[0190] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 110-111, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 107-108. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 110-111. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 110-111. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 110-111. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NOs: 110-111. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NOs: 16-18, 33, 34, 49, or 50. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 107-108. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 107-108. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 107-108. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NO: 107-108. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NOs: 13-15, 31, 32, 47, or 48.

[0191] In some embodiments, the TCR can comprise a beta chain construct and an alpha chain construct. In some embodiments, the TCR beta chain construct of the engineered immune cell can comprise a complementarity determining region 3 (CDR3). In some embodiments, the CDR3 can have an amino acid sequence CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the recombinant nucleic acid encodes a TCR that recognizes an HLA-A02:01 epitope (e.g., an epitope having an amino acid sequence as set forth in SEQ ID NO: 116). In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSVWASGGYEQYF (SEQ ID NO: 105). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence as set forth in any one of SEQ ID NOs: 6, 24, 40, 58-80. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 114. In some embodiments, the TCR beta chain construct comprises a complementaritydetermining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 103. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 104.

[0192] In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:100. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein theCDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:101. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%,at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence CAGLADYGGSQGNLIF (SEQ ID NO: 102).

[0193] In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 113. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 106. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 112.

[0194] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 119-120, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 117-118. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 119-120. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 119-120. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 119-120. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NOs: 119-120. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NOs: 16-18, 33, 34, 49, or 50. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 117-118. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 117-118. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 117-118. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NO: 117-118. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NOs: 13-15, 31, 32, 47, or 48.

[0195] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 126, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 125.

[0196] Provided herein is an isolated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct; wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A11:01 allele. The epitope can comprise a RAS G12V mutation. The epitope can comprise a sequence of VVGAVGVGK. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSVDSSHNEQFF (SEQ ID NO: 6). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 90% sequence identityto an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

[0197] In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5.

[0198] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%,at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

[0199] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 16-18, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 13-15. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 16-18. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 16-18. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 16-18. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NOs: 16-18. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 13-15. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 13-15. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 13- 15. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NOs: 13-15.

[0200] Provided herein is an isolated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one TCR comprising a TCR alpha chain construct and / or a TCR beta chain construct; wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-C01:02 allele. The epitope can comprise a RAS G12V mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSRSWPPGYTF (SEQ ID NO: 24). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSRSWPPGYTF (SEQ ID NO: 24). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSRSWPPGYTF (SEQ ID NO: 24). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence as set forth in CASSRSWPPGYTF (SEQ ID NO: 24). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to CASSRSWPPGYTF (SEQ ID NO: 24). In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 28-30. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 28-30. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 28-30. In some embodiments, the TCR beta chain construct comprises avariable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 28-30. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 28-30. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 22. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 23.

[0201] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%,at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 21.

[0202] In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 27.

[0203] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 33-34, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 31-32. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 33-34. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 33-34. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 33-34. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NOs: 33-34. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 31-32. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 31-32. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 31- 32. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NOs: 31-32.

[0204] In some embodiments, the TCR binds to a complex comprising (i) the epitope from human RAS comprising the mutation G12V and (ii) an MHC protein encoded by an HLA- C01:02 allele. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASRDMTGKAFF (SEQ ID NO: 40). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASRDMTGKAFF (SEQ ID NO: 40). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequencewith at least 95% sequence identity to CASRDMTGKAFF (SEQ ID NO: 40). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASRDMTGKAFF (SEQ ID NO: 40). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASRDMTGKAFF (SEQ ID NO: 40). In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 44-46. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 44-46. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 44-46. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 44-46. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 44-46. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 38. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 39.

[0205] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 35. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 36. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 43.

[0206] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth inSEQ ID NO: 47-48. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NO: 47-48.

[0207] In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:35. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein theCDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO:36. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%,at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 43. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 43.

[0208] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth inSEQ ID NOs: 47-48. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NOs: 49-50. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NOs: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NOs: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NOs: 47-48. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with 100% identity to an amino acid sequence as set forth in SEQ ID NOs: 47-48.

[0209] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-A68:01 allele, wherein the mutated epitope from human RAS is characterized by a G 12V mutation. The epitope can comprise a sequence of VVGAVGVGK. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSLSLNTEAFF (SEQ ID NO: 132). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSLSLNTEAFF (SEQ ID NO: 132). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSLSLNTEAFF (SEQ ID NO: 132). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSLSLNTEAFF (SEQ ID NO: 132). In some embodiments, the TCR beta chainconstruct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSLSLNTEAFF (SEQ ID NO: 132).

[0210] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 130. In some embodiments, the TCR beta chain construct comprises a complementaritydetermining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 131 or a combination thereof.

[0211] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 127. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 128. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 129. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acidsequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 133. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 133. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 133. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 133. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 133.

[0212] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele with a KD of at most 1000 nM, wherein the mutated epitope from human RAS is characterized by a G12D mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele with a KD of 1000 nM or more, wherein the mutated epitope from human RAS is characterized by a G12D mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASTWGEGEAFF (SEQ ID NO: 140). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASTWGEGEAFF (SEQ ID NO: 140). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASTWGEGEAFF (SEQ ID NO: 140). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASTWGEGEAFF (SEQ ID NO: 140). In someembodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to CASTWGEGEAFF (SEQ ID NO: 140).

[0213] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 138. In some embodiments, the TCR beta chain construct comprises a complementaritydetermining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 139 or a combination thereof.

[0214] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 135. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 136. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 137. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acidsequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 141. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 141. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 141. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 141. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 141.

[0215] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically can bind to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele. The mutated epitope from human RAS can be characterized by a G12D mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSPGSSYEQYF (SEQ ID NO: 148). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSPGSSYEQYF (SEQ ID NO: 148). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSPGSSYEQYF (SEQ ID NO: 148). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSPGSSYEQYF (SEQ ID NO: 148). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSPGSSYEQYF (SEQ ID NO: 148).

[0216] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 146. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 147.

[0217] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 143. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 144. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 145. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 149. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 149. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identityto an amino acid sequence as set forth in SEQ ID NO: 149. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 149. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 149.

[0218] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR can specifically bind to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C05:01 allele. The mutated epitope from human RAS can be characterized by a G12D mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSFFLGETQYF (SEQ ID NO: 156). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSFFLGETQYF (SEQ ID NO: 156). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSFFLGETQYF (SEQ ID NO: 156). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSFFLGETQYF (SEQ ID NO: 156). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to CASSFFLGETQYF (SEQ ID NO: 156).

[0219] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as setforth in SEQ ID NO: 158. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 158. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 158. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 158. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 158. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 154. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 155 or a combination thereof.

[0220] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has anamino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 151. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 152. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 153. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 157. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 157. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 157. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 157. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%,at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 157.

[0221] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele and a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSLPRMGSDTEAFF (SEQ ID NO: 164). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSEPRMGSDTEAFF (SEQ ID NO: 164). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSLPRMGSDTEAFF (SEQ ID NO: 164). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSLPRMGSDTEAFF (SEQ ID NO: 164). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSLPRMGSDTEAFF (SEQ ID NO: 164).

[0222] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 162. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 163 or a combination thereof.

[0223] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 159. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 160. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 161. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 165. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 165. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identityto an amino acid sequence as set forth in SEQ ID NO: 165. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 165. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 165.

[0224] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR can specifically bind to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele. The mutated epitope from human RAS can be characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR can specifically bind to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele. The mutated epitope from human RAS can be characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR can specifically bind to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele and a human MHC encoded by an HLA- C03:04 allele. The mutated epitope from human RAS can be characterized by a G12V mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSPTRLGYNEQFF (SEQ ID NO: 172). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSPTRLGYNEQFF (SEQ ID NO: 172). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSPTRLGYNEQFF (SEQ ID NO: 172). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSPTRLGYNEQFF (SEQ ID NO: 172). In some embodiments, the TCR beta chain construct comprises a complementarity determining region3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to CASSPTRLGYNEQFF (SEQ ID NO: 172).

[0225] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 170. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%,at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 171 or a combination thereof.

[0226] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 167. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 168. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 169. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ IDNO: 173. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 173. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 173. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 173. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 173.

[0227] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele and a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSLRTGEAFF (SEQ ID NO: 180). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSLRTGEAFF (SEQ ID NO: 180). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequencewith at least 95% sequence identity to CASSLRTGEAFF (SEQ ID NO: 180). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSLRTGEAFF (SEQ ID NO: 180). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to CASSLRTGEAFF (SEQ ID NO: 180).

[0228] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 182. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 182. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 182. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 182. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 182. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 178. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 179 or a combination thereof.

[0229] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 175. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 176. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 177. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 181. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 181. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 181. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 181. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 181.

[0230] Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. Provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) comprising a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR specifically binds to a mutated epitope from human RAS in complex with a human MHC encoded by an HLA-CO3:O3 allele and a human MHC encoded by an HLA-C03:04 allele, wherein the mutated epitope from human RAS is characterized by a G12V mutation. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acidsequence with at least 80% sequence identity to CASSIDYRGIGETQYF (SEQ ID NO: 188). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSIDYRGIGETQYF (SEQ ID NO: 188). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSIDYRGIGETQYF (SEQ ID NO: 188). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSIDYRGIGETQYF (SEQ ID NO: 188). In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to CASSIDYRGIGETQYF (SEQ ID NO: 188).

[0231] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 190. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 190. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 190. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 190. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 190. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 186. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 187 or a combination thereof.

[0232] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 183. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 184. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%,at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 185. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 189. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 189. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 189. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 189. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 189.

[0233] In some embodiments, the TCR binds to a complex comprising (i) the epitope from human RAS comprising the mutation G12D and (ii) an MHC protein encoded by an HLA- A11:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to CASSSSGGRIYGYTF (SEQ ID NO: 196). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 90% sequence identity to CASSSSGGRIYGYTF (SEQ ID NO: 196). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 95% sequence identity to CASSSSGGRIYGYTF (SEQ ID NO: 196). In some embodiments, the TCR beta chain construct comprises a CDR3 having an amino acid sequence with 100% sequence identity to CASSSSGGRIYGYTF (SEQ ID NO: 196). In someembodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to CASSSSGGRIYGYTF (SEQ ID NO: 196).

[0234] In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 198. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 198. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 198. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 198. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 198. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 194. In some embodiments, the TCR beta chain construct comprises a complementaritydetermining region 2 (CDR2) having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 195 or a combination thereof.

[0235] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a CDR1, wherein the CDR1 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 191. In some embodiments, the TCR alpha chain construct comprises a CDR2, wherein the CDR2 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 192. In some embodiments, the TCR alpha chain construct comprises a CDR3, wherein the CDR3 has an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 193. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acidsequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 197. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 197. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 197. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 197. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 197.

[0236] In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, atleast 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the TCR can comprise a CDR3 with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 80.

[0237] In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the TCR can comprise a G12V 9mer with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 67.

[0238] Provided herein is an isolated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct; wherein the TCR specifically bindsto an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. The epitope can comprise a RAS G12V mutation. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NO: 408, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 411, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 411. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 411. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 411. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 411. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 411.

[0239] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 408. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 408. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 408. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 408. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 408.

[0240] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 413, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 412. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 413. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 413. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 413. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 413. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 412. In someembodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 412. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 412. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 412.

[0241] Provided herein is an isolated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct; wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. The epitope can comprise a RAS G12V mutation. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 424, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 427, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 425-427. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 427. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 427. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 427. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 427.

[0242] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 422-424. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 424. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 424. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 424. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 424.

[0243] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 429, and / or (b) an alpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 428. In some embodiments, the TCR comprises a beta chain having an aminoacid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 429. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 429. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 429. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 429. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 428. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 428. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 428. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 428.

[0244] Provided herein is an isolated nucleic acid or a cell comprising a recombinant nucleic acid, wherein the nucleic acid encodes at least one T cell receptor (TCR) comprising a TCR alpha chain construct and / or a TCR beta chain construct; wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. The epitope can comprise a RAS G12V mutation. In some embodiments, the TCR alpha chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 391, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR beta chain construct comprises a variable region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 394, wherein the TCR specifically binds to an epitope from RAS in complex with a human MHC encoded by an HLA-A03:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 392-394. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 394. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 394. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 394. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 394.

[0245] In some embodiments, the TCR comprises a TCR alpha chain construct. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 391. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 391. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 391. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with at least 95% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 391. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence with 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 391.

[0246] In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 396, and / or (b) analpha chain having an amino acid sequence with at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 395. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 396. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 396. In some embodiments, the TCR comprises a beta chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 396. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 396. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 80% identity to an amino acid sequence as set forth in SEQ ID NO: 395. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 395. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 395. In some embodiments, t...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered immune cell comprising: a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; and a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed.

2. The engineered immune cell of claim 1, wherein the engineered immune cell further comprises an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell.

3. The engineered immune cell of claim 2, wherein the exogenous TCR molecule binds to the exogenous peptide when presented in complex with the MHC.

4. An engineered immune cell comprising: a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule; and an exogenous peptide presented in complex with an MHC on the surface of the engineered immune cell, wherein the exogenous TCR molecule binds to the exogenous peptide when presented in complex with the MHC.

5. The engineered immune cell of claim 4, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed.

6. The engineered immune cell of any one of claims 1-5, wherein the exogenous co-stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

7. The engineered immune cell of any one of claims 1-6, wherein activation of the exogenous co-stimulatory receptor molecule provides a co-stimulatory signal to the engineered immune cell.

8. The engineered immune cell of any one of claims 1-7, wherein the exogenous co-stimulatory receptor molecule or the surface protein comprises one or more proteinsselected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL.

9. The engineered immune cell of any one of claims 1-8, wherein the exogenous co-stimulatory receptor molecule or the surface protein comprises CD70 and / or CD80.

10. The engineered immune cell of any one of claims 1-8, wherein the exogenous co-stimulatory receptor molecule or the surface protein comprises CD70 and CD80.

11. The engineered immune cell of any one of claims 1-8, wherein the exogenous co-stimulatory receptor molecule or the surface protein comprises LIGHT.

12. The engineered immune cell of any one of claims 1-8, wherein the exogenous co-stimulatory receptor molecule or the surface protein comprises CD86.

13. The engineered immune cell of any one of claims 1-12, wherein the engineered immune cell further comprises a third nucleic acid molecule encoding a molecule having transposase activity.

14. The engineered immune cell of claim 13, wherein the molecule is a transposase.

15. The engineered immune cell of claim 14, wherein the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity.

16. The engineered immune cell of claim 15, wherein the transposase is Sleeping Beauty transpose SB100X.

17. The engineered immune cell of any one of claims 1-16, wherein the first nucleic acid molecule comprises a DNA.

18. The engineered immune cell of claim 17, wherein the DNA is a DNA nanoplasmid comprising a transposon.

19. The engineered immune cell of claim 18, wherein the exogenous TCR molecule is stably expressed in the engineered immune cell.

20. The engineered immune cell of any one of claims 1-19, wherein the second nucleic acid molecule comprises an RNA.

21. The engineered immune cell of any one of claims 1-20, wherein the exogenous co-stimulatory molecule is transiently expressed in the immune cell.

22. The engineered immune cell of any one of claims 13-21, wherein the third nucleic acid molecule comprises an RNA.

23. The engineered immune cell of any one of claims 1-22, wherein the first nucleic acid molecule further encodes an enhancer.

24. The engineered immune cell of claim 23, wherein the enhancer comprises a switch receptor.

25. The engineered immune cell of claim 24, wherein the switch receptor is a PD 1-4 IBB or a IL2 receptor.

26. The engineered immune cell of claim 25, wherein the IL2 receptor comprises a mutation.

27. The engineered immune cell of any one of claims 1-26, wherein the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a ligand for the exogenous co-stimulatory receptor molecule.

28. The engineered immune cell of claim 27, wherein the ligand is CD27.

29. The engineered immune cell of claim 27, wherein the ligand is CD28 or CTLA4.

30. The engineered immune cell of any one of claims 27-29, wherein the ligand is an exogenous ligand or an endogenous ligand.

31. The engineered immune cell of any one of claims 1-30, wherein the first nucleic acid molecule or the second nucleic acid molecule is delivered into the engineered immune cell via an electroporation or a particle.

32. The engineered immune cell of claim 31, wherein the particle is a polyalkyleneimine or a lipid.

33. The engineered immune cell of claim 32, wherein the particle is a lipid with a cationic headgroup.

34. The engineered immune cell of claim 33, wherein the particle comprises a pH responsive lipid.

35. The engineered immune cell of claim 34, wherein the particle comprises a PEGylated-lipid.

36. The engineered immune cell of claim 35, wherein the particle is a lipid particular, polymer particle, or mixture thereof.

37. The engineered immune cell of any one of claims 31-36, wherein the particle is a nanoparticle.

38. The engineered immune cell of any one of claims 31-37, wherein the particle is a lipid nanoparticle (LPD), a lipoplex (LPX), a polyplex (PLX), or a lipopolyplex (LPLX) particle.

39. The engineered immune cell of any one of claims 31-38, wherein the particle further comprises at least one phosphatidylserine.

40. The engineered immune cell of any one of claims 31-39, wherein the particles are nanoparticles, in which:(i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and / or(ii) the nanoparticles have a neutral or net negative charge and / or(iii) the zeta potential of the nanoparticles is 0 or less.

41. The engineered immune cell of any one of claims 31-40, wherein the particle comprises polyalkyleneimine.

42. The engineered immune cell of claim 41, wherein (a) the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) is 2.0 to 15.0; or (b) the molar ratio of the number of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the first, second, or the third nucleic acid molecule (N:P ratio) is at least about 48.

43. The engineered immune cell of claim 42, wherein the ionic strength of the composition is about 50 mM or less.

44. The engineered immune cell of claim 43, wherein the concentration of monovalent cationic ions is about 25 mM or less and the concentration of divalent cationic ions is about 20 pM or less.

45. The engineered immune cell of any one of claims 31-44, wherein the particle is a polyplex particle.

46. The engineered immune cell of any one of claims 31-45, wherein the particle comprises a hydrophobic moiety having a binding moiety covalently attached thereto.

47. The engineered immune cell of claim 46, wherein the hydrophobic moiety having a binding moiety covalently attached thereto and the particle are non-covalently associated with each other.

48. The engineered immune cell of claim 46 or 47, wherein the hydrophobic moiety having a binding moiety covalently attached thereto is an integral part of the particle.

49. The engineered immune cell of any one of claims 46-48, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a polymer.

50. The engineered immune cell of any one of claims 46-49, wherein the hydrophobic moiety having the binding moiety covalently attached thereto comprises a compound of Formula IL-X1-P-X2-B (I) whereinP comprises a polymer;L comprises a hydrophobic moiety attached to a first end of the polymer;B comprises a binding moiety attached to a second end of the polymer;XI is absent or a first linking moiety; andX2 is absent or a second linking moiety.

51. The engineered immune cell of claim 50, wherein XI comprises a carbonyl group.

52. The engineered immune cell of claim 50 or 51, wherein X2 comprises the reaction product of a maleimide group with a thiol or cysteine group of a compound comprising the binding moiety.

53. The engineered immune cell of any one of claims 46-52, wherein the hydrophobic moiety is or is comprised in a lipid.

54. The engineered immune cell of any one of claims 49-53, wherein the polymer provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion.

55. The engineered immune cell of any one of claims 49-54, wherein the polymer comprises polyethylene glycol (PEG).

56. The engineered immune cell of any one of claims 46-55, wherein the hydrophobic moiety having a binding moiety covalently attached thereto comprises a compound of Formula IIwherein B comprises the binding moiety.

57. The engineered immune cell of claim 56, wherein B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.

58. The engineered immune cell of any one of claims 46-57, wherein the binding moiety covalently attached to the hydrophobic moiety comprises an antibody or an antibody derivative.

59. The engineered immune cell of any one of claims 31-58, wherein the particle is complexed with the nucleic acid molecules and / or encapsulates the nucleic acid molecules.

60. The engineered immune cell of any one of claims 1-59, wherein the engineered immune cell is administered to a subject in need thereof.

61. The engineered immune cell of claim 60, wherein the subject has cancer.

62. The engineered immune cell of any one of claims 2-61, wherein the engineered immune cell has been incubated with the exogenous peptide.

63. The engineered immune cell of claim 62, wherein the engineered immune cell has been activated in the presence of the exogenous peptide in complex with the MHC molecule.

64. The engineered immune cell of claim 63, wherein the exogenous peptide is a T cell epitope.

65. The engineered immune cell of claim 64, wherein the T cell epitope is from a PRAME protein.

66. The engineered immune cell of claim 65, wherein the T cell epitope comprises an amino acid sequence of SLLQHLIGL (SEQ ID NO: 116).

67. The engineered immune cell of claim 66, wherein the MHC molecule is HLA 02:01.

68. The engineered immune cell of claim 64, wherein the T cell epitope is from a RAS protein.

69. The engineered immune cell of claim 68, wherein the RAS protein is a KRAS protein.

70. The engineered immune cell of claim 69, wherein the KRAS protein comprises a mutation.

71. The engineered immune cell of claim 70, wherein the mutation is a G12V mutation, a G12D mutation, or a G12C mutation.

72. The engineered immune cell of any one of claims 68-71, wherein the T cell epitope comprises an amino acid sequence selected from the group consisting of VVGAVGVGK (SED ID NO: 51), VVVGAVGVGK (SED ID NO: 52), AVGVGKSAL (SED ID NO: 53), GADGVGKSAL (SED ID NO: 54), GAVGVGKSAL (SED ID NO: 55), GAVGVGKSA (SED ID NO: 56), and VVVGADGVGK(SED ID NO: 57).

73. The engineered immune cell of claim 72, wherein the MHC molecule is HLA Al 1:01, HLA A03:01, HLA A68:01, HLA C0L02, HLA C03:03 / C03:04, HLA C05:01, orHLA A1 L01.

74. The engineered immune cell of claim 73, wherein the peptide is not processed by the engineered immune cell.

75. The engineered immune cell of any one of claims 1-74, wherein the first nucleic acid molecule comprises a promoter selected from the group consisting of EFl alpha, EFl alpha- Human T-lymphotropic virus 1 (HTLV), MP71, and MP71-HTLV.

76. The engineered immune cell of claim 75, wherein the promoter is EFlalpha.

77. The engineered immune cell of claim 75, wherein the promoter is EFlalpha-HTLV.

78. The engineered immune cell of claim 75, wherein the promoter is MP71.

79. The engineered immune cell of claim 75, wherein the promoter is MP71-HTLV.

80. The engineered immune cell of claim 75, wherein the promoter is MP71- HTLV and co-electroporated with the first nucleic acid molecule.

81. The engineered immune cell of any one of claims 1-80, wherein the first nucleic acid molecule is a TCR that recognizes an epitope from a PRAME protein in complex with an MHC molecule encoded by an HLA A02:01 allele.

82. The engineered immune cell of any one of claims 1-81, wherein the TCR binds to a PRAME epitope in complex with an MHC encoded by an HLA A02:01 allele.

83. The engineered immune cell of claim 82, wherein the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

84. The engineered immune cell of any one of claims 1-83, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105.

85. The engineered immune cell of claim 84, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115.

86. The engineered immune cell of claim 84 or 85, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104.

87. The engineered immune cell of any one of claims 84-86, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102.

88. The engineered immune cell of any one of claims 84-87, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113.

89. The engineered immune cell of any one of claims 84-88, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

90. The engineered immune cell of any one of claims 84-89, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

91. The engineered immune cell of any one of claims 1-80, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA Al 1:01 allele.

92. The engineered immune cell of any one of claims 1-91, wherein the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by an HLA Al 1:01 allele.

93. The engineered immune cell of claim 92, wherein the TCR binds to the epitope comprising an amino acid sequence of VVGAVGVGK (SEQ ID NO: 51) in complex with an MHC encoded by an HLA Al 1:01 allele.

94. The engineered immune cell of any one of claims 1-80 or 91-93, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6.

95. The engineered immune cell of claim 94, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

96. The engineered immune cell of claim 94 or 95, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

97. The engineered immune cell of any one of claims 94-96, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3.

98. The engineered immune cell of any one of claims 94-97, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

99. The engineered immune cell of any one of claims 94-98, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

100. The engineered immune cell of any one of claims 94-99, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

101. The engineered immune cell of any one of claims 1-100, wherein the engineered immune cell further comprises a fourth nucleic acid molecule encoding the exogenous TCR for transiently expressing the exogenous TCR.

102. The engineered immune cell of claim 101, wherein the fourth nucleic acid molecule comprises an RNA.

103. The engineered immune cell of claim 101 or 102, wherein the exogenous TCR transiently expressed in the engineered immune cell increases cell expansion.

104. The engineered immune cell of claims 101-103, wherein the engineered immune cell is a population of engineered immune cells, and wherein, after being incubated for a period time, the number of the population of engineered immune cells is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more higher than the number ofan otherwise identical population of engineered immune cells without the fourth nucleic acid molecule.

105. The engineered immune cell of claim 104, wherein the population of engineered immune cells have been incubated for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days or more.

106. The engineered immune cell of claim 104, wherein the population of engineered immune cells have been incubated for no more than 15 days, no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day or less.

107. The engineered immune cell of claim 106, wherein the population of engineered immune cells have been incubated in the presence of a cytokine.

108. The engineered immune cell of claim 107, wherein the cytokine is IL-7 and / or IL-15.

109. The engineered immune cell of any one of claims 1-108, wherein the engineered immune cell retains TCR expression and / or proliferative capacity with or without cryopreservation.

110. The engineered immune cell of claim 109, wherein the engineered immune cell post cryopreservation exhibits comparable TCR expression and / or proliferative capacity compared to an otherwise identical engineered immune cell without cryopreservation.

111. The engineered immune cell of any one of claims 1-110, wherein the engineered immune cell exhibits comparable or increased cytotoxicity towards tumor cells than an otherwise identical cell being retrovirally engineered.

112. The engineered immune cell of any one of claims 1-111, wherein the engineered immune cell retains comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution.

113. The engineered immune cell of any one of claims 1-112, wherein the engineered immune cell retains comparable expansion capacity after being diluted by 1: 10 and 1:20.

114. The engineered immune cell of any one of claims 1-113, wherein the engineered immune cell is administered into a subject in need thereof after being incubated ex vivo for no more than 15 days, no more than 14 days, no more than 13 days, no more than 12 days, no more than 11 days, no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day or less.

115. The engineered immune cell of claim 114, wherein the population of engineered immune cells have been incubated in the presence of a cytokine.

116. The engineered immune cell of claim 115, wherein the cytokine is IL-7 and / or IL-15.

117. A cell culture comprising: a population of immune cells comprising a plurality of engineered immune cells comprising a first engineered immune cell and a second engineered immune cell, wherein the first engineered immune cell of the plurality comprises a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR, and wherein the first and / or the second engineered immune cell of the plurality comprises a second nucleic acid molecule encoding an exogenous co- stimulatory molecule comprising a second nucleotide sequence encoding an exogenous co- stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or (ii) the exogenous costimulatory receptor molecule is transiently expressed; and a peptide, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells for stimulating the first engineered immune cell.

118. The cell culture of claim 117, wherein the peptide is presented on a surface protein of an engineered immune cell and / or a non-engineered immune cell of the population of immune cells.

119. The cell culture of claim 117 or 118, wherein the peptide is presented on a surface protein of a non-engineered immune cell of the population of immune cells.

120. The cell culture of any one of claims 117-119, wherein the peptide is presented on a surface protein of an engineered immune cell of the population of immune cells.

121. The cell culture of any one of claims 117-120, wherein the peptide is presented on a surface protein of the second engineered immune cell of the plurality.

122. The cell culture of any one of claims 117-121, wherein the peptide is presented on an MHC molecule of an immune cell of the population of immune cells.

123. The cell culture of any one of claims 117-122, wherein the peptide is a T cell epitope.

124. The cell culture of any one of claims 117-123, wherein the exogenous costimulatory receptor molecule is transiently expressed.

125. The cell culture of any one of claims 117-124, wherein the exogenous costimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

126. The cell culture of any one of claims 117-125, wherein activation of the exogenous co-stimulatory receptor molecule provides a co- stimulatory signal to the engineered immune cell.

127. The cell culture of any one of claims 117-126, wherein the exogenous costimulatory receptor molecule or the surface protein comprises one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL.

128. The cell culture of any one of claims 117-127, wherein the exogenous costimulatory receptor molecule or the surface protein comprises CD70 and / or CD80.

129. The cell culture of any one of claims 117-127, wherein the exogenous costimulatory receptor molecule or the surface protein comprises CD70 and CD80.

130. The cell culture of any one of claims 117-127, wherein the exogenous costimulatory receptor molecule or the surface protein comprises LIGHT.

131. The cell culture of any one of claims 117-127, wherein the exogenous costimulatory receptor molecule or the surface protein comprises CD86.

132. The cell culture of any one of claims 117-131, wherein the engineered immune cell further comprises a third nucleic acid molecule encoding a molecule having transposase activity.

133. The cell culture of claim 132, wherein the molecule is a transposase.

134. The cell culture of claim 133, wherein the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity.

135. The cell culture of claim 134, wherein the transposase is Sleeping Beauty transpose SB100X.

136. The cell culture of any one of claims 117-135, wherein the first nucleic acid molecule comprises a DNA.

137. The cell culture of claim 136, wherein the DNA is a DNA nanoplasmid comprising a transposon.

138. The cell culture of claim 137, wherein the exogenous TCR is stably expressed in the immune cell.

139. The cell culture of any one of claims 117-138, wherein the second nucleic acid molecule comprises an RNA.

140. The cell culture of any one of claims 117-139, wherein the exogenous costimulatory molecule is transiently expressed in the immune cell.

141. The cell culture of any one of claims 132-140, wherein the third nucleic acid molecule comprises an RNA.

142. The cell culture of any one of claims 117-141, wherein the first nucleic acid molecule further encodes an enhancer.

143. The cell culture of claim 142, wherein the enhancer comprises a switch receptor.

144. The cell culture of claim 143, wherein the switch receptor is a PD1-41BB or a IL2 receptor.

145. The cell culture of claim 144, wherein the IL2 receptor comprises a mutation.

146. The cell culture of any one of claims 117-145, wherein the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a ligand for the exogenous co- stimulatory receptor molecule.

147. The cell culture of claim 146, wherein the ligand is CD27.

148. The cell culture of claim 146, wherein the ligand is CD28 or CTLA4.

149. The cell culture of any one of claims 117-148, wherein the first nucleic acid molecule or the second nucleic acid molecule is delivered into the engineered immune cell via electroporation or a particle.

150. A method for producing an antigen specific T cell, the method comprising:(a) providing a population of immune cells comprising a plurality of engineered immune cells comprising a first engineered immune cell and a second engineered immune cell, wherein the first engineered immune cell of the plurality comprises a first nucleic acid encoding an exogenous TCR, and wherein the first and / or the second engineered immune cell of the plurality comprises a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotidesequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or (ii) the exogenous co- stimulatory receptor molecule is transiently expressed; and(b) culturing the population of immune cells in the presence of a peptide, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells, thereby stimulating the first engineered immune cell and producing the antigen specific T cell.

151. The method of claim 150, wherein the culturing is no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days or less.

152. The method of claim 150 or 151, wherein the culturing is no more than 3 days.

153. The method of any one of claims 150-152, wherein the culturing is no more than 2 days.

154. A method for producing an antigen specific T cell, the method comprising:(a) providing a plurality of engineered immune cells;(b) culturing the plurality of engineered immune cells ex vivo to generate a therapeutically effective amount of antigen specific T cells, wherein the culturing is less than 7 days; and(c) administering the plurality of engineered immune cells into a subject in need thereof.

155. The method of claim 154, wherein the plurality of engineered immune cells comprises a first engineered immune cell and a second engineered immune cell, each engineered immune cell of the plurality comprises a first nucleic acid encoding an exogenous TCR, a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed.

156. The method of claim 154 or 155, wherein culturing in (b) comprises culturing the plurality of engineered immune cells in the presence of a peptide.

157. The method of claim 156, wherein the peptide is presented on the surface protein of the second engineered immune cell for stimulating the first engineered immune cell.

158. The method of any one of claims 154-157, wherein the therapeutically effective amount of antigen specific T cells comprises at least 104T cells.

159. The method of any one of claims 154-158, wherein providing the plurality of engineered immune cells comprises: providing a population of immune cells from a subject; and delivering into the population of immune cells the first nucleic acid encoding the exogenous TCR, and the second nucleic acid encoding the exogenous co-stimulatory molecule, thereby generating the plurality of engineered immune cells.

160. The method of any one of claims 154-159, wherein the culturing is less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days or less.

161. The method of any one of claims 154-160, wherein the exogenous costimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

162. The method of any one of claims 154-161, wherein each engineered immune cell of the plurality further comprises a third nucleic acid encoding a transposase.

163. The method of any one of claims 154-162, further comprising, prior to delivering, selecting CD4+ T cells and / or CD8+ T cells from the population of immune cells.

164. The method of any one of claims 154-163, wherein the population of immune cells are isolated from a sample from the subject.

165. The method of claim 164, wherein the sample is a blood sample or a PBMC sample.

166. The method of any one of claims 154-165, wherein delivering comprises electroporating the first, the second, and / or the third nucleic acid molecule into the population of immune cells.

167. The method of claim 166, wherein delivering comprises using a particle to deliver the first, the second, and / or the third nucleic acid molecule into the population of immune cells.

168. The method of claim 167, wherein the particle is a lipid nanoparticle (LNP), a lipoplex (LPX), a polyplex (PLX), a lipopolyplex (LPLX) particle, or any combination thereof.

169. The method of any one of claims 150-168, wherein the peptide is a T cell epitope.

170. The method of claim 169, wherein the peptide is not further processed to be presented in complex with an MHC molecule.

171. The method of claim 170, wherein the antigen specific T cell comprises a plurality of antigen specific T cells.

172. The method of claim 171, wherein the plurality of antigen specific T cells retain TCR expression and / or proliferative capacity with or without cryopreservation.

173. The method of claim 172, wherein the plurality of antigen specific T cells post cry opreservation exhibits comparable TCR expression and / or proliferative capacity compared to an otherwise identical plurality of antigen specific T cells without cryopreservation.

174. The method of claim 172 or 173, wherein further comprising administering the plurality of antigen specific T cells into a subject in need thereof.

175. The method of any one of claims 172-174, wherein further comprising administering the plurality of antigen specific T cells into a subject in need thereof after no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days of culturing.

176. The method of any one of claims 172-175, wherein further comprising administering the plurality of antigen specific T cells into a subject in need thereof, and wherein the plurality of antigen specific T cells have been cryopreserved after no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days of culturing.

177. The method of any one of claims 174-176, wherein the plurality of antigen specific T cells expand in the subject.

178. The method of claim 177, wherein the plurality of antigen specific T cells expand better than retrovirally engineered T cells, and wherein the number of the plurality of antigen specific T cells after expansion is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or more, higher than the retrovirally engineered T cells after expansion when the same amount of cells are administered into the subject.

179. The method of any one of claims 172-178, wherein the plurality of antigen specific T cells exhibit comparable or increased cytotoxicity towards tumor cells than retrovirally engineered T cells.

180. The method of any one of claims 1-179, wherein the engineered immune cell retains comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution.

181. The engineered immune cell of any one of claims 1-180, wherein the engineered immune cell retains comparable expansion capacity after being diluted by 1:10 and 1:20.

182. The method of any one of claims 150-180, wherein the TCR binds to a PRAME epitope in complex with an MHC encoded by an HLA 02:01 allele.

183. The method of claim 182, wherein the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

184. The method of any one of claims 150-183, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105.

185. The method of claim 184, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115.

186. The method of claim 184 or 185, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104.

187. The method of any one of claims 184-186, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102.

188. The method of any one of claims 184-187, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113.

189. The method of any one of claims 184-188, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

190. The method of any one of claims 184-189, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

191. The method of any one of claims 150-180, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA Al 1:01 allele.

192. The method of any one of claims 150-180, wherein the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by an HLA Al 1:01 allele.

193. The method of claim 192, wherein the TCR binds to the epitope comprising an amino acid sequence of VVGAVGVGK (SEQ ID NO: 51) in complex with an MHC encoded by an HLA Al 1:01 allele.

194. The method of any one of claims 150-180 or 191-192, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6.

195. The method of claim 194, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

196. The method of claim 194 or 195, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

197. The method of any one of claims 194-196, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3.

198. The method of any one of claims 194-197, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

199. The method of any one of claims 194-198, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

200. The method of any one of claims 194-199, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

201. A method of treating a cancer in a subject in need thereof, the method comprising:(a) administering an ex vivo population of T cells comprising antigemMHC complex activated T cells, wherein the ex vivo population of T cells comprises at most 108T cells, wherein the antigemMHC complex activated T cells expand in vivo in the subject to at least 2-fold after administration to the subject or expand in vivo to at least IO10cells after administration to the subject.

202. The method of claim 201, wherein further comprising, prior to (a), culturing the ex vivo population of T cells in the presence of an antigemMHC complex.

203. The method of claim 202, wherein T cells are autologous T cells.

204. The method of claim 203, wherein the ex vivo population of T cells are cultured ex vivo for no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days or less.

205. The method of any one of claims 201-204, wherein the T cells comprises an engineered immune cell, wherein the engineered immune cell comprises a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR molecule; and a second nucleic acid molecule comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule.

206. The method of claim 205, wherein (i) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the engineered immune cell and / or (ii) the exogenous co-stimulatory receptor molecule is transiently expressed.

207. The method of claim 205 or 206, wherein the exogenous co-stimulatory receptor molecule is a surface protein endogenously expressed on an antigen presenting cell (APC).

208. The method of any one of claims 205-207, wherein activation of the exogenous co-stimulatory receptor molecule provides a co-stimulatory signal to the engineered immune cell.

209. The method of any one of claims 205-208, wherein the exogenous co- stimulatory receptor molecule or the surface protein comprises one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL.

210. The method of any one of claims 205-209, wherein the exogenous co- stimulatory receptor molecule or the surface protein comprises CD70 and / or CD80.

211. The method of claim any one of claims 205-209, wherein the exogenous costimulatory receptor molecule or the surface protein comprises CD70 and CD80.

212. The method of claim any one of claims 205-209, wherein the exogenous costimulatory receptor molecule or the surface protein comprises LIGHT.

213. The method of claim any one of claims 205-209, wherein the exogenous costimulatory receptor molecule or the surface protein comprises CD86.

214. The method of any one of claims 205-213, wherein the engineered immune cell further comprises a third nucleic acid molecule encoding a transposase.

215. The method of claim 214, wherein the transposase is Sleeping Beauty, PiggyBac, Frog, Prince, Himarl, Passport, Minos, hAT, Toll, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, Hsmarl, or a functionally equivalent variant thereof having transposase / transposition activity.

216. The method of claim 215, wherein the transposase is Sleeping Beauty transpose SB100X.

217. The method of any one of claims 205-216, wherein the first nucleic acid molecule comprises a DNA.

218. The method of claim 217, wherein the DNA is a DNA nanoplasmid comprising a transposon.

219. The method of claim 218, wherein the exogenous TCR is stably expressed in the immune cell.

220. The method of any one of claims 205-219, wherein the second nucleic acid molecule comprises an RNA.

221. The method of claim any one of claims 205-220, wherein the exogenous costimulatory molecule is transiently expressed in the immune cell.

222. The method of claim any one of claims 205-221, wherein the third nucleic acid molecule comprises an RNA.

223. The method of claim any one of claims 205-222, wherein the first nucleic acid molecule further encodes an enhancer.

224. The method of claim 223, wherein the enhancer comprises a switch receptor.

225. The method of claim 224, wherein the switch receptor is a PD1-41BB or a IL2 receptor.

226. The method of claim 225, wherein the IL2 receptor comprises a mutation.

227. The method of any one of claims 206-226, wherein the engineered immune cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an exogenous ligand for the exogenous co-stimulatory receptor molecule.

228. The method of claim 227, wherein the exogenous ligand is CD27.

229. The method of claim 227, wherein the exogenous ligand is CD28 or CTLA4.

230. The method of any one of claims 201-229, wherein the antigen is a cancer antigen.

231. The method of any one of claims 201-230, wherein the antigen comprises a mutation.

232. The method of any one of claims 201-231, wherein the T cells are produced by the method of any one of claims 150-200.

233. A method for producing a population of immune cells comprising a plurality of antigen specific T cells, the method comprising:(a) providing a population of at least IxlO9immune cells;(b) introducing into immune cells of the population of immune cells a first nucleic acid encoding an exogenous TCR and a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co-stimulatory receptor molecule, thereby producing a population of immune cells comprising a plurality of engineered T cells, wherein the plurality of engineered T cells comprises a first engineered immune cell and a second engineered immune cell, wherein(i) the first engineered immune cell comprises the first nucleic acid,(ii) the first and / or the second engineered immune cell comprises the second nucleic acid, and(iii) the second nucleotide sequence is not integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cell and / or(iv) the exogenous co-stimulatory receptor molecule is transiently expressed;(c) expanding the population of immune cells comprising the plurality of engineered T cells in the presence of a peptide for a first time period of 2 days, wherein the peptide is presented on a surface protein of an immune cell of the population of immune cells comprising the plurality of engineered T cells and a complex comprising the surface protein and the peptide is recognized by the an exogenous TCR, thereby expanding the first engineered immune cell and producing the population of immune cells comprising a plurality of antigen specific T cells, wherein(d) the percentage of T cells comprising the first nucleic acid is at least 10% of the total number of T cells in the population of immune cells at the first time period, and / or(e) when the population of immune cells comprising the plurality of engineered T cells is expanded in the presence of the peptide for a second time period of 5 or more days the percentage of T cells comprising the first nucleic acid is at least 50% of the total number of T cells in the population of immune cells at the second time period.

234. The method of claim 233, wherein providing in (a) comprises providing a population of at least 2xl09immune cells.

235. The method of any one of claims 233 or 234, wherein after (b) and before (c), the population of immune cells comprising a plurality of engineered T cells comprises less than 20% viable immune cells.

236. The method of any one of claims 233-235, wherein expanding in (c) comprises preferentially expanding the first engineered immune cell.

237. The method of any one of claims 233-236, wherein immune cells without the first nucleic acid are not substantially expanded in step (c).

238. An ex vivo activated and / or expanded population of T cells produced by the method of any one of claims 150-200.

239. The ex vivo activated and / or expanded population of T cells of claim 238, wherein the ex vivo activated and / or expanded population of T cells, are cultured for no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days.

240. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-116, and a pharmaceutically acceptable carrier.

241. Use of a plurality of T cells for treating a cancer in a subject in need thereof, comprising administering at most 106T cells, wherein the T cells have been activated ex vivo in the present of an antigen in complex with an MHC molecule, wherein the T cells expand in vivo in the subject to at least 2-fold or at least 1010cells.

242. Use of the ex vivo activated and / or expanded population of T cells of claim 238 or 240, the engineered immune cell of any one of claims 1-116, or the engineered immune cell of claim 240 in the manufacture of a medicament for treating cancer.

243. The use of claim 241 or 242, wherein the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

244. The use of claim 243, wherein the cancer is selected from the group consisting of bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, carcinoma of the sexual and reproductive organs, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors glioma, meningioma, and pituitary adenoma.

245. The use of claim 244, wherein the cancer is a solid cancer.

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