WO2025224684A1 - Compositions and methods for producing engineered immune cells - Google Patents

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 IMMUNE

CEEES

CROSS-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; B comprises 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 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).

[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% 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.

[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 immune cell 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 a genomic 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 10⁴ T 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 some embodiments, 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 and a 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 10⁸ T 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 10¹⁰ cells 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 exogenous co-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 IxlO⁹ immune 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 2xl0⁹ immune 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 10⁶ T 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 10¹⁰ cells.

[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 by reference 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, RV TCR-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 INVENTION

Definitions

[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. K_off 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). K_on refers 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 comprising CD8+ 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, 8^TH Ed., Lange Publishing, Los Altos, Calif. (1994). For a detailed description of the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3^rd Ed., 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 amino acid 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 compared and 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 encodes at 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 surface protein. 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 have amino 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 exogenous ligand 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 be non-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 acid molecule 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 domain sequences (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 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 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 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 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: 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 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: 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 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: 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 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:

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).

[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%, 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: 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%, 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 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 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: 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 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: 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 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: 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 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:

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 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 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 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: 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 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: 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 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: 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%, 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: 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 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 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 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: 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 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: 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 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: 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 sequence with 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 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: 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 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: 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 in SEQ 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 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.

[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%, 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: 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 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 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 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: 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 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: 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 acid sequence 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 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 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 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: 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 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: 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 acid sequence 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 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 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 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: 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 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 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 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 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 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 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 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: 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 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: 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 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: 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 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 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 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 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 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: 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 ID NO: 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 sequence with 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 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: 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%, 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: 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 acid sequence 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 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 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 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: 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%, 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: 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 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 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 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: 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 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: 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 acid sequence 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%, 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: 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 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: 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 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: 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%, 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: 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 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: 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 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: 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%, at least 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 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: 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 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: 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 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: 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 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: 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 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: 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 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: 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 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: 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 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: 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 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: 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 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 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 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: 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 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: 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 amino acid 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) 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: 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, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 395.

[0247] 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 G12D 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: 295, 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 selected from SEQ ID NO: 298, 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 NO: 298. 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: 298. 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: 298. 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: 298. 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: 298.

[0248] 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: 295. 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 Nos: 295. 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: 295. 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: 295. 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: 295. [0249] 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: 300, 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: 299. 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: 300. 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: 300. 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: 300. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 300. 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: 299. 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: 299. 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: 299. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 299.

[0250] 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 G12D 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: 311, 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: 314, 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 NO: 314. 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: 314. 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: 314. 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: 314. 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: 314.

[0251] 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: 311. 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: 311. 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: 311. 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: 311. 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: 311.

[0252] 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: 316, 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: 315. 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: 316. 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: 316. 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: 316. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 316. 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: 315. 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: 315. 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: 315. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 315.

[0253] 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 G12D 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: 327, 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 selected from SEQ ID NO: 330, 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 NO: 330. 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: 330. 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: 330. 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: 330. 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: 330.

[0254] 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: 327. 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: 327. 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: 327. 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: 327. 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: 327.

[0255] 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: 332, 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: 331. 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: 332. 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: 332. 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: 332. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 332. 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: 331. 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: 331. 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: 331. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 331.

[0256] 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 G12D 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: 343, 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: 346, 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 NO: 346. 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: 346. 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: 346. 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: 346. 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: 346.

[0257] 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: 343. 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: 343. 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: 343. 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: 343. 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: 343.

[0258] 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: 348, 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: 347. 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: 348. 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: 348. 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: 348. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO: 348. 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: 347. 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: 347. 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: 347. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 347.

[0259] 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 G12D 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: 359, 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 selected from SEQ ID NO: 362, 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 NO: 362. 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: 362. 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: 362. 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: 362. n 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: 362.

[0260] 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: 359. 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: 359. 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: 359. 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: 359. 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: 359.

[0261] 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: 364, 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: 363. 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: 364. 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: 364. 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: 364. In some embodiments, the TCR comprises a beta chain having an amino acid sequence as set forth in SEQ ID NO64. 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: 363. 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: 363. 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: 363. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence as set forth in SEQ ID NO: 363.

[0262] The engineered immune cell may have been incubated with an exogenous peptide. The engineered immune cell may have been activated in the presence of the exogenous peptide in a complex with the MHC molecule. The exogenous peptide can be a T cell epitope. The T cell epitope can be from a PRAME protein or a RAS protein. The T cell epitope can comprise an amino acid sequence of SLLQHLIGL (SEQ ID NO: 116).

[0263] The engineered immune cell can comprise the MHC molecule may be HLA 02 :01. The engineered immune cell can comprise the T cell epitope may be from a RAS protein. The RAS protein can be a KRAS protein. In some cases, the KRAS protein can comprise a mutation. In some cases, the mutation may be a G12V mutation, a G12D mutation, or a G12C mutation. The T cell epitope can comprise 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). The MHC molecule can comprise HLA All:01, HLA A03:01, HLA A68:01, HLA C0E02, HLA C03:03/C03:04, HLA C05:01, or HLA Al 1:01.

[0264] The engineered immune cell can comprise a peptide. In some cases, the peptide may not be processed by the engineered immune cell.

[0265] The engineered immune cell can comprise the first nucleic acid molecule. The first nucleic acid molecule can comprise a promoter selected from the group consisting of EFl alpha, EFl alpha- Human T-lymphotropic virus 1 (HTLV), MP71, and MP71-HTLV. In some cases, the promoter may be EFl alpha. In some cases, the promoter may be EFl alpha- HTLV. In some cases, the promoter may be MP71. In some cases, the promoter may be MP71-HTLV. In some cases, the promoter may be MP71-HTLV and co-electroporated with the first nucleic acid molecule.

[0266] The first nucleic acid molecule can be a TCR that recognizes an epitope from a PRAME protein in a complex with an MHC molecule encoded by an HLA A02:01 allele.

[0267] The engineered immune cell can comprise TCR that may bind to a PRAME epitope in complex with an MHC encoded by an HLA A02:01 allele. In some cases, the PRAME epitope can comprise an amino acid sequence of SEQ ID NO: 116.

[0268] A TCR may comprise a TCR beta chain construct and a TCR alpha chain construct. In some cases, the TCR beta chain construct can comprise a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105. In some cases, the TCR beta chain construct can comprise 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.

[0269] The TCR beta chain construct can comprise 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.

[0270] The TCR alpha construct can comprise a CDR1, a CDR2, and a CDR3. In some cases, the CDR1 may have an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 may have an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 may have an amino acid sequence set forth in SEQ ID NO: 102. [0271] The TCR alpha chain construct can comprise 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.The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 124. In some cases, TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 124. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123. In some cases, TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 123.

[0272] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 126. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 126. In some cases, the TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125. In some cases, the TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 125.

[0273] The TCR can bind to a complex comprising an epitope from human RAS comprising a mutation G12V. In some cases, the TCR binds to a complex that may comprise an MHC protein encoded by an HLA Al 1:01 allele. The TCR can bind 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. The TCR can bind 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.

[0274] The TCR can comprise a TCR beta chain construct and a TCR alpha chain construct. In some cases, the TCR beta chain construct can comprise a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6. The TCR beta chain construct can comprise 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. The TCR beta chain construct can comprise a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4. In some cases, the TCR beta chain construct can comprise a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

[0275] The TCR alpha chain construct can comprise a CDR1, a CDR2, and a CDR3. In some cases, the CDR1 may have an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 may have an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 may have an amino acid sequence set forth in SEQ ID NO: 3. The TCR alpha chain construct can comprise 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.

[0276] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 16. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 16. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13. In some cases, TCR can comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

[0277] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 17. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 17. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14. In some cases, TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 14.

[0278] The engineered immune cell can further comprise a fourth nucleic acid molecule encoding the exogenous TCR. In some cases, the engineered immune cell comprising the exogenous TCR can be transiently expressed. The fourth nucleic acid molecule can comprise an RNA. In some cases, the exogenous TCR transiently expressed in the engineered immune cell may increase cell expansion.

[0279] The engineered immune cell comprises a population of engineered immune cells, and wherein, after being incubated for a period time, the number of the population of engineered immune cells may be 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. The population of engineered immune cells may 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. The population of engineered immune cells may 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. The population of engineered immune cells may have been incubated in the presence of a cytokine. The cytokine may be IL-7 and/or IL- 15. Cryopreservation

[0280] Cryopreservation may enable the long-term storage of cells. Cryopreservation may be utilized when there is a surplus of cells available for subculturing. Cryopreservation may require that cells be treated with an appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below -130°C until they are needed.

[0281] The engineered immune cell may retain TCR expression and/or proliferative capacity with or without cryopreservation. The engineered immune cell post cryopreservation may exhibit comparable TCR expression and/or proliferative capacity compared to an otherwise identical engineered immune cell without cryopreservation. The engineered immune cell may exhibit comparable or increased cytotoxicity towards tumor cells than an otherwise identical cell being retrovirally engineered. The engineered immune cell may retain comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution. The engineered immune cell may retain comparable expansion capacity after being diluted by 1 : 10. In some cases, the engineered immune cell may retain comparable expansion capacity after being diluted by 1:20. In some cases, the engineered immune cells may retain comparable expansion capacity after being diluted by 1:5, by 1:6, by 1:7, by 1:8, by 1:9, by 1: 10, by 1: 11, by 1: 12, by 1: 13, by 1: 14, by 1: 15, by 1: 16, by 1: 17, by 1: 18, by 1: 19, or by 1:20. The engineered immune cell may be 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. The population of engineered immune cells may have been incubated in the presence of a cytokine. The cytokine may be IL-7 and/or may be IL- 15.

Cell Cultures

[0282] The present disclosure provides compositions and methods for culturing the engineered immune cells.

[0283] Cell culture techniques may be utilized to generate a population of immune cells. Cell culture may be used for drug screening and development, and large-scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins).

[0284] Cell culture may refer to the growth of cells under controlled conditions within the laboratory (i.e., in vitro). Cell culture may refer to the removal of cells from an animal and/or plant and their subsequent growth in a favorable artificial environment. The cells may be removed directly from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.

[0285] Cells may consist of primary culture. Primary culture can refer to the stage of the culture after the cells are isolated from the issue and proliferate under the appropriate conditions until they occupy all of the available substrate (z.e., reach confluence). At this stage, the cells may have to be passaged (z.e., subculture) by transferring them to a new well with fresh growth medium to provide more room for continued growth.

[0286] Growth medium or culture medium can be a solid, liquid or semi- solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation. Different types of media may be used for growing different types of cells.

[0287] The cell culture described herein can comprise a population of immune cells comprising a plurality of engineered immune cells. The plurality of engineered immune cells can comprise a first engineered immune cell and a second engineered immune cell. The first engineered immune cell of the plurality can comprise a first nucleic acid molecule comprising a first nucleotide sequence encoding an exogenous TCR. In some cases, the first and/or the second engineered immune cell of the plurality can comprise a second nucleic acid molecule encoding an exogenous co-stimulatory molecule. The exogenous co- stimulatory molecule can comprise 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 first engineered immune cell nor the second engineered immune cell. In some cases, the exogenous co-stimulatory receptor molecule can be transiently expressed. In some cases, the first and/or the second engineered immune cell of the plurality can further comprise a peptide. The peptide may be presented on a surface protein of an immune cell of the population of immune cells for stimulating the first engineered immune cell. The peptide can be 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 cases, the peptide may be presented on a surface protein of a non-engineered immune cell of the population of immune cells. In some cases, the peptide may be presented on a surface protein of an engineered immune cell of the population of immune cells. In some cases, the peptide may be presented on a surface protein of the second engineered immune cell of the plurality. In some cases, the peptide may be presented on an MHC molecule of an immune cell of the population of immune cells. The peptide can be a T cell epitope. In some cases, the exogenous co-stimulatory receptor molecule can be transiently expressed. The exogenous co-stimulatory receptor molecule may be a surface protein endogenously expressed on an antigen presenting cell (APC). The activation of the exogenous co-stimulatory receptor molecule may provide a co-stimulatory signal to the engineered immune cell.

[0288] The exogenous co-stimulatory receptor molecule or the surface protein may comprise one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and/or CD80. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and CD80. The exogenous co-stimulatory receptor molecule or the surface protein can comprise LIGHT. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD86.

[0289] The engineered immune cell can further comprise a third nucleic acid molecule encoding a molecule having transposase activity. The molecule may be a transposase. The transposase may be 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 may be Sleeping Beauty transpose SB 100X.

[0290] The first nucleic acid molecule can comprise a DNA. The DNA can be a DNA nanoplasmid comprising a transposon. The exogenous TCR may be stably expressed in the immune cell. The second nucleic acid molecule can comprise an RNA. In some cases, the exogenous co-stimulatory molecule can be transiently expressed in the immune cell. The third nucleic acid molecule can comprise an RNA. The first nucleic acid molecule can further encode an enhancer. The enhancer can comprise a switch receptor. The switch receptor can be a PD1- 4 IBB receptor. In some cases, the switch receptor can be a IL2 receptor. The IL2 receptor can comprise a mutation.

[0291] The cell culture can comprise an engineered immune cell further comprising a nucleic acid molecule. The nucleic acid molecule can comprise a nucleotide sequence encoding a ligand for the exogenous co-stimulatory receptor molecule. The ligand may be CD27. The ligand may be CD28. The ligand may be CTLA4. The first nucleic acid molecule may be delivered into the engineered immune cell via electroporation or a particle. The second nucleic acid molecule may be delivered into the engineered immune cell via electroporation or a particle.

Methods of Producing Engineered Immune Cells

[0292] The present disclosure provides methods described herein (e.g., Insta TCR process) to produce the engineered immune cells. The methods can be used to provide a therapeutically effective amount of antigen specific T cells within a short period of time (e.g., less than 10 days, less than 8 days, less than 5 days, less than 3 days, or less than 2 days).

[0293] The method for producing an antigen specific T cell can comprise providing a population of immune cells comprising a plurality of engineered immune cells. The plurality of engineered immune cells can comprise a first engineered immune cell and a second engineered immune cell. The first engineered immune cell of the plurality can comprise a first nucleic acid encoding an exogenous TCR. The first and/or the second engineered immune cell of the plurality can comprise a second nucleic acid. The second nucleic acid can comprise 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 first engineered immune cell nor the second engineered immune cell. In some cases, the exogenous co- stimulatory receptor molecule may be transiently expressed. In some cases, culturing the population of immune cells can be the presence of a peptide. The peptide can be presented on the 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. The culturing may be 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. The culturing may be no more than 3 days. The culturing may be no more than 2 days.

[0294] The method for producing an antigen specific T cell can comprise providing a plurality of engineered immune cells. The plurality of engineered immune cells can be isolated from a subject. The method can further comprise culturing the plurality of engineered immune cells ex vivo to generate a therapeutically effective amount of antigen specific T cells. The method can further comprise culturing the plurality of engineered immune cells for less than 7 days. In some cases, the method can further comprise culturing the plurality of engineered immune cells for less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days or less than 2 days. The method can further comprise administering the plurality of engineered immune cells into a subject in need thereof. [0295] The plurality of engineered immune cells can comprise a first engineered immune cell and a second engineered immune cell. Each engineered immune cell of the plurality can comprise 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. 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 not be transiently expressed. The method can further comprise 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, in the presence of a peptide. The peptide may be presented on the surface protein of the second engineered immune cell for stimulating the first engineered immune cell.

[0296] The method can comprise producing a therapeutically effective amount of antigen specific T cells comprising at least 10⁴ T cells. In some cases, the method can comprise producing a therapeutically effective amount of antigen specific T cells comprising at least 10³ T cells, at least 10⁵ T cells, at least 10⁶ T cells, at least 10⁷ T cells, at least 10⁸ T cells, at least 10⁹ T cells, at least IO¹⁰ T cells, at least 10¹¹ T cells, at least 10¹² T cells, at least 10¹³ T cells, at least 10¹⁴ T cells, at least 10¹⁵ T cells, at least 10¹⁶ T cells, at least 10¹⁷ T cells, at least 10¹⁸ T cells, at least 10¹⁹ T cells, or at least IO²⁰ T cells. The method can further comprise providing the plurality of engineered immune cells comprising 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. The method can comprise culturing the plurality of engineered immune cells for less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days or less. The exogenous co- stimulatory receptor molecule may be a surface protein endogenously expressed on an antigen presenting cell (APC).

[0297] Each engineered immune cell of the plurality can further comprise a third nucleic acid encoding a transposase. The transposase may be 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. The transposase may be Sleeping Beauty transpose SB100X.

[0298] The method can further comprise, prior to delivering, selecting CD4+ T cells and/or CD8+ T cells from the population of immune cells. The population of immune cells may be isolated from a sample from the subject. The sample may be a blood sample or a PBMC sample. [0299] The method for delivering can further comprise electroporating the first, the second, and/or the third nucleic acid molecule into the population of immune cells. Delivering can comprise using a particle to deliver the first, the second, and/or the third nucleic acid molecule into the population of immune cells. The particle may be a lipid nanoparticle (ENP), a lipoplex (EPX), a polyplex (PEX), a lipopolyplex (EPEX) particle, or any combination thereof. [0300] The peptide may be a T cell epitope. The peptide may not be further processed to be presented in complex with an MHC molecule. The antigen specific T cell can comprise a plurality of antigen specific T cells. The plurality of antigen specific T cells can retain TCR expression and/or proliferative capacity with or without cryopreservation. The plurality of antigen specific T cells post cryopreservation can exhibit comparable TCR expression and/or proliferative capacity compared to an otherwise identical plurality of antigen specific T cells without cryopreservation.

[0301] The plurality of antigen specific T cells produced by the methods described herein can be administered into a subject in need thereof. The administration of the plurality of antigen specific T cells into a subject in need thereof can be performed after no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days after culturing. The plurality of antigen specific T cells can further be administered 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. The plurality of antigen specific T cells may expand in the subject. The plurality of antigen specific T cells may expand better than retrovirally engineered T cells or other conventional methods known in the art. In some cases, the number of the plurality of antigen specific T cells after expansion may comprise 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. The plurality of antigen specific T cells produced by the methods described herein may exhibit comparable or increased cytotoxicity towards tumor cells than retrovirally engineered T cells.

[0302] The engineered immune cell produced by the methods described herein may retain comparable expansion capacity after being diluted or being administered into a subject compared to an otherwise identical engineered immune cell without dilution. In some cases, the engineered immune cells may retain comparable expansion capacity after being diluted by 1: 10 and 1:20. In some cases, the engineered immune cells may retain comparable expansion capacity after being diluted by 1:5, by 1:6, by 1:7, by 1:8, by 1:9, by 1: 10, by 1: 11, by 1: 12, by 1: 13, by 1: 14, by 1: 15, by 1: 16, by 1: 17, by 1: 18, by 1: 19, or by 1:20.

[0303] The antigen specific immune cells produced by the methods can comprise a TCR binding to a PRAME epitope in complex with an MHC encoded by an HLA 02:01 allele. In some cases, the PRAME epitope can comprise an amino acid sequence of SEQ ID NO: 116. [0304] A TCR may comprise a TCR beta chain construct and a TCR alpha chain construct. In some cases, the TCR beta chain construct can comprise a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105. In some cases, the TCR beta chain construct can comprise 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.

[0305] The TCR beta chain construct can comprise 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. [0306] The TCR alpha construct can comprise a CDR1, a CDR2, and a CDR3. In some cases, the CDR1 may have an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 may have an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 may have an amino acid sequence set forth in SEQ ID NO: 102.

[0307] The TCR alpha chain construct can comprise 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.

[0308] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 124. In some cases, TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 124. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123. In some cases, TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 123.

[0309] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 126. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 126. In some cases, the TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125. In some cases, the TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 125.

[0310] The TCR can bind to a complex comprising an epitope from human RAS comprising a mutation G12V. In some cases, the TCR binds to a complex that may comprise an MHC protein encoded by an HLA Al 1:01 allele. The TCR can bind 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. The TCR can bind 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. [0311] The TCR can comprise a TCR beta chain construct and a TCR alpha chain construct. In some cases, the TCR beta chain construct can comprise a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6. The TCR beta chain construct can comprise 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. The TCR beta chain construct can comprise a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4. In some cases, the TCR beta chain construct can comprise a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

[0312] The TCR alpha chain construct can comprise a CDR1, a CDR2, and a CDR3. In some cases, the CDR1 may have an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 may have an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 may have an amino acid sequence set forth in SEQ ID NO: 3. The TCR alpha chain construct can comprise 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.

[0313] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 16. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 16. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13. In some cases, TCR can comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

[0314] The TCR can comprise a beta chain having an amino acid sequence set forth in SEQ ID NO: 17. In some cases, the TCR can comprise a beta chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 17. In some cases, TCR can comprise an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14. In some cases, TCR can comprise an alpha chain having an amino acid sequence that may be at least 80% identical to SEQ ID NO: 14.

Methods of Treatment

[0315] The present disclosure described herein provides methods of treatment using the engineered immune cells (e.g., manufactured through the Insta TCR process).

[0316] The method of treatment described herein can comprise treating a cancer in a subject thereof. The method can comprise administering an ex vivo population of T cells comprising the antigemMHC complex activated T cells, wherein the ex vivo population of T cells comprises at most 10⁸ T cells. In some cases, the population of T cells can comprise at most 10⁹ T cells, at most 10¹⁰ T cells, at most 10¹¹ T cells, at most 10¹² T cells, at most 10¹³ T cells, at most 10¹⁴ T cells, at most 10¹⁵ T cells, at most 10¹⁶ T cells, at most 10¹⁷ T cells, at most IO¹⁸ T cells, at most 10¹⁹ T cells, or at most IO²⁰ T cells. The method can further comprise the antigen: MHC complex activated T cells expanded in vivo in the subject to at least 2-fold after administration to the subject or expand in vivo to at least IO¹⁰ cells after administration to the subject. In some cases, the antigemMHC complex activated T cells may expand in vivo in the subject to at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold after administration to the subject. In some cases, the antigen: MHC complex activated T cells expand in vivo in the subject to at least 10⁹ cells, at least IO¹⁰ cells, at least 10¹¹ cells, at least 10¹² cells, at least 10¹³ cells, at least 10¹⁴ cells, at least 10¹⁵ cells, at least 10¹⁶ cells, least most 10¹⁷ cells, at least 10¹⁸ cells, at least 10¹⁹ cells, or at least IO²⁰ cells after administration to the subject. The method can further comprise, prior to administering the ex vivo population of T cells, culturing the ex vivo population of T cells in the presence of an antigen: MHC complex.

[0317] In some cases, the method of producing the ex vivo population of T cells can comprise providing a plurality of cells a subject. The plurality of cells can be delivered with one or more nucleic acid molecules described herein by electroporation. The electroporation may reduce live cell percentage of the plurality of cells. In some cases, the plurality of cells after electroporation can comprise at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, or at most 70% live cells. The live cells of the plurality of cells can expand after culturing using the methods provided herein. The plurality of cells after culturing or expansion can comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% live cells. [0318] T cells may be autologous T cells. Autologous T cells can be isolated from the subject that will be administered with the ex vivo activated T cells. T cells may be allogeneic T cells.

[0319] The ex vivo population of T cells may be 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. In some cases, 10% of the ex vivo population of T cells may be antigen specific T cells on day 3 of culture. In some cases, over 70% of the ex vivo population of T cells may be antigen specific T cells after activation by co-stimulatory molecules CD80 and CD70. In some cases, costimulatory molecules may be CD70 or CD80. In some cases, over 70% of the ex vivo population of T cells may be antigen specific T cells on day 5, on day 7, on day 9 and on day 10. In some cases, antigen specific T cells TCR cells may keep increasing in culture.

[0320] The T cells can comprise 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. 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 not be transiently expressed. The exogenous costimulatory receptor molecule may be a surface protein endogenously expressed on an antigen presenting cell (APC). In some cases, activation of the exogenous co-stimulatory receptor molecule may provide a co-stimulatory signal to the engineered immune cell. The exogenous co-stimulatory receptor molecule or the surface protein can comprise one or more proteins selected from the group consisting of CD70, CD80, LIGHT, CD86, 41BBL, CD58, and ICOSL. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and/or CD80. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD70 and CD80. The exogenous co-stimulatory receptor molecule or the surface protein can comprise LIGHT. The exogenous co-stimulatory receptor molecule or the surface protein can comprise CD86.

[0321] The engineered immune cell can further comprise a third nucleic acid molecule encoding a transposase. The transposase can be 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. The transposase can be Sleeping Beauty transpose SB100X.

[0322] The first nucleic acid molecule can comprise a DNA. DNA can be a DNA nanoplasmid comprising a transposon. The exogenous TCR can be stably expressed in the immune cell. The second nucleic acid molecule can comprise an RNA. In some cases, the exogenous co- stimulatory molecule may be transiently expressed in the immune cell. The third nucleic acid molecule can comprise an RNA.

[0323] The first nucleic acid molecule can further encode an enhancer. The enhancer can comprise a switch receptor. The switch receptor can be a PD1-41BB receptor. In some cases, the switch receptor can be an IL2 receptor. The IL2 receptor can comprise a mutation.

[0324] The engineered immune cell can further comprise a nucleic acid molecule. In some cases, the nucleic acid molecules can comprise a nucleotide sequence encoding an exogenous ligand for the exogenous co-stimulatory receptor molecule. The exogenous ligand may be CD27. The exogenous ligand may be CD28. The exogenous ligand may be CTLA4.

[0325] The antigen may be a cancer antigen. The antigen may comprise a mutation.

[0326] The methods of treatment using the engineered immune cells can comprise the T cells produced by the methods of producing the engineered immune cell. [0327] The method for producing a population of immune cells can comprise a plurality of antigen specific T cells comprising providing a population of at least IxlO⁹ immune cells. In some cases, the population of immune cells can be at least IxlO⁷ immune cells, IxlO⁸ immune cells, IxlO⁹ immune cells, IxlO¹⁰ immune cells, IxlO¹¹ immune cells, or IxlO¹² immune cells. The method can further comprise introducing into the population of immune cells a first nucleic acid encoding an exogenous and a second nucleic acid comprising a second nucleotide sequence encoding an exogenous co -stimulatory receptor molecule. In some cases, the population of immune cells can comprise a plurality of engineered T cells comprising a first engineered immune cell and a second engineered immune cell. The method can further comprise a first engineered immune cell comprising the first nucleic acid, the first and/or the second engineered immune cell comprising the second nucleic acid, and the second nucleotide sequence not being integrated into a genomic nucleic acid molecule of the first engineered immune cell nor the second engineered immune cells. In some cases, the exogenous costimulatory receptor molecule may be transiently expressed.

[0328] The method can further comprise 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. In some cases, the first time period can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 20 days or more. The peptide may be presented on the 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 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. The method may further comprise the percentage of T cells comprising the first nucleic acid to be at least 10% of the total number of T cells in the population of immune cells at the first time period. The method may further comprise when the population of immune cells comprising the plurality of engineered T cells may be expanded in the presence of the peptide for a second time period of 5 or more days (e.g., 6 days, 7 days, 8 days, 9 days, 10 days, 20 days or more) and the percentage of T cells can comprise 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. In some cases, the total number of T cells in the population of immune cells at the second time period can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. The method can further comprise providing at least 2xl0⁹ immune cells. The plurality of engineered immune cells, prior to expanding the population of immune cells comprising the plurality of engineered T cells, can further comprise less than 20% viable immune cells. In some cases, the plurality of engineered T cells can comprise less than 10%, can comprise less than 20%, can comprise less than 30% or can comprise less than 40% after electroporation. The method can further comprise, while expanding the population of immune cells comprising the plurality of engineered T cells, preferentially expanding the first engineered immune cell. In some cases, immune cells without the first nucleic acid may not be substantially expanded.

[0329] An ex vivo activated and/or expanded population of T cells may be produced by the method of culturing engineered immune cell. The ex vivo activated and/or expanded population of T cells may be cultured for no more than 10 days, no more than 5 days, no more than 4 days, or no more than 2 days.

[0330] A pharmaceutical composition may comprise an engineered immune cell, and a pharmaceutically acceptable carrier.

[0331] Use of a plurality of T cells for treating a cancer in a subject in need thereof, can comprise administering at most 10⁶ T cells. The T cells may have been activated ex vivo in the present of an antigen in complex with an MHC molecule. In some cases, the T cells expand in vivo in the subject to at least 2-fold or at least 10¹⁰ cells. In some cases, the T cells expand in vivo in the subject to at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 10,000-fold, at least 100,000-fold or more. In some cases, the T cells expand in vivo in the subject to at least 2⁹ cells, at least 10¹⁰, at least 10¹¹, at least 10¹², at least 10¹³, at least 10¹⁴, at least 10¹⁵, at least 10¹⁶, at least 10¹⁷, at least 10¹⁸, at least 10¹⁹, at least IO²⁰, at least 10²¹, at least 10²² or more. The present disclosure provides the use of the ex vivo activated and/or expanded population of T cells of the plurality of T cells or pharmaceutical composition, or the engineered immune, in the manufacture of a medicament for treating cancer. The cancer may be carcinoma, lymphoma, blastoma, sarcoma, or leukemia. The cancer may be 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. The cancer may be a solid cancer.

T-cell receptors

[0332] The engineered cells provided herein can comprise a recombinant nucleic acid encoding a TCR that recognizes epitopes. The T-cell receptor (TCR) can comprise a TCR beta chain construct and a TCR alpha chain construct.

[0333] In some embodiments, the population of T cells expressing the TCR specific to the peptide:MHC complex are primary T cells. In some embodiments, the population of T cells expressing the TCR specific to the peptide:MHC complex is a cell line. In some embodiments, the cell line is a Jurkat cell line.

[0334] In some embodiments, the T cell can be a genetically-modified cell comprising in its genome a modified human T-cell receptor (TCR) alpha chain gene and/or a modified human TCR beta chain gene, wherein the cell has reduced cell-surface expression of the endogenous TCR.

[0335] Gene-editing nucleases can be employed in order to disrupt components of the TCR. The TCR alpha chain (TCRa) is encoded by a single TRAC gene and pairs with the TCR beta chain (TCRP) encoded by two TCRB genes. Since the TCR a/p dimer can produce a fully functioning TCR complex, disrupting TCRa and/or TCRP function can reduce (even eliminate) endogenous TCR expression.

[0336] Various methods can be used to disrupt endogenous TCRa or TCRP genes. For example, four classes of gene editing proteins exist that share a common mode of action in binding a user defined sequence of DNA and mediating a double stranded DNA break (DSB). Zinc finger nucleases (ZFN) are heterodimeric arrays that co-localize at a target DNA site. ZFNs include individual finger subunits that bind DNA and are tethered to the Fokl nuclease domain that cleaves DNA. Transcription activator-like effector nucleases (TALEN) include repeating units that bind DNA by virtue of a hypervariable two amino acid sequence (repeat variable di-residue; RVD) that governs DNA base recognition. Similar to ZFNS, TALENs function as dimeric proteins that are fused to the Fokl endonuclease domain for DSB generation. Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site. The clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platform involves a small guide RNA (gRNA) transcript that contacts a target DNA sequence via Watson-Crick base pairing and the Cas9 nuclease that cleaves the DNA. [0337] In some embodiments, introducing the genome-editing nuclease into the T cell includes introducing into the T cell a polynucleotide that encodes the genome-editing nuclease.

[0338] In some embodiments, introducing the genome-editing nuclease into the T cell includes introducing into the T cell a Cas9 polypeptide. In some embodiments, the genome-editing nuclease includes a TALEN nuclease, a CRISPR/Cas9 nuclease, or a megaTAL nuclease.

[0339] In some embodiments, the CRISPR/Cas9 nuclease is derived from either Streptococcus pyogenes or Staphylococcus aureus. In some of these embodiments, the CRISPR/Cas9 nuclease includes a nuclease-resistant gRNA such as, for example, at least one 2’-OMe- phosphorothioate modified base, at least one 2’-O-methyl modified base, or at least one 2’-O- methyl 3’ thioPACE modified base.

[0340] In some embodiments, the TALEN nuclease or the megaTAL nuclease is encoded by an RNA that has an exogenous polyadenylation signal.

[0341] In some embodiments, the method described herein can further include culturing the T cell under conditions effective for expanding the population of genome-modified T cells.

[0342] In some embodiments, disrupting expression of TCRa and/or TCRP further disrupts assembly of TCRa and TCRp. In some embodiments, disrupting expression of TCRa further disrupts formation of a complex between TCR and CD3. In some embodiments, disrupting expression of TCRa involves further disrupting assembly of TCRa and TCRp.

[0343] In some embodiments, a genetically-modified T cell comprises a disrupted TCR alpha chain and/or beta chain and an inactivated gene encoding immune checkpoint protein such as PD1 and CTLA-4. This can be made possible by gene inactivation using specific TALE- nucleases directed against TCRalpha or TCRbeta coupled with inactivation of genes encoding immune checkpoint protein such as PD1 and CTLA-4.

[0344] In some embodiments, the genetic modification relies on the inactivation of one gene, or two genes selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha and TCR beta. In some embodiments, the genetic modification relies on the inactivation of two genes selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG 3 and TCR alpha, LAG 3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCRbeta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, 2B4 and TCR beta. In some embodiments, the genetic modification relies on the inactivation of more than two genes. The genetic modification can be operated ex- vivo.

[0345] The ability of T cells to recognize antigens 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 diversity of the TCR. This multi-subunit immune recognition receptor can associate with the CD3 complex and bind peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The first signal in activation of T cells can be provided by binding of the T-cell receptor to a short peptide presented by the MHC on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually an antigen-presenting cell such as a professional antigen-presenting cell, B-cells and macrophages can be important APCs. Binding of a TCR to the antigenic peptide on the APC can be a central event in T cell activation, which occurs at an immunological synapse at the point of contact between the T cell and the APC.

[0346] Each TCR can comprise variable complementarity determining regions (CDRs), as well as framework regions (FRs) and a constant region. The TCR described herein may be in soluble form. The TCR described herein may not comprise a constant region. The TCR described herein may comprise a functional fragment of a full-length TCR. The TCR described herein may comprise the variable region of the alpha chain or beta chain only. The amino acid sequence of the third complementarity-determining region (CDR3) loops of the alpha (or a) and beta (or P) chain variable domains largely determines the sequence diversity of aP T cells arising from recombination between variable (VP), diversity (DP), and joining (JP) gene segments in the P chain locus, and between analogous Va and Ja gene segments in the a chain locus, respectively. The existence of multiple such gene segments in the TCR a and P chain loci allows for a large number of distinct CDR3 sequences to be encoded. Independent addition and deletion of nucleotides at the VP-DP, Dp-jp, and Va-Ja junctions during the process of TCR gene rearrangement further increases CDR3 sequence diversity. In this respect, immunocompetence is reflected in the diversity of TCRs. The yS TCR is distinctive from the aP TCR in that it encodes a receptor that interacts closely with the innate immune system. TCRyS, is expressed early in development, has specialized anatomical distribution, unique small-molecule specificities, and has a broad spectrum of innate and adaptive cellular interactions. Early in ontogeny, as the restricted subsets of TCRyS cells populate various tissues prenatally, a biased pattern of TCRy V and J segment expression is established. [0347] TCRs can bind to a peptide: MHC complex. In some cases, the TCR can bind to an MHC peptide complex comprising an epitope from an antigen or candidate antigen described herein. In some cases, the TCR can bind to an MHC peptide complex. The binding of the TCR complex and the epitope bound with the MHC molecule (e.g., MHC class I or MHC class II) can trigger signal transduction pathways that lead to regulation of immune responses against the antigenic peptide. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of 1 pM to 1 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 500 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 250 nM. In some embodiments, the TCR binds to an MHC- peptide complex with a KD or an IC50 of less than 150 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 100 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 50 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 25 nM. In some embodiments, the TCR binds to an MHC-peptide complex with a KD or an IC50 of less than 10 nM.

[0348] In some embodiments, the nucleic acid is operably linked to a promoter. In various embodiments, the nucleic acid sequence encoding a TCR is codon optimized.

[0349] In some embodiments, the TCR sequence is comprised of human sequences. In some embodiments, the TCR sequence is comprised of non-human animal sequences that have been humanized using any method of humanization known in the art. The TCRs provided herein can be engineered TCRs.

[0350] In some embodiments, the tumor cell line, SNG-M, can endogenously express HLA- Al l:01 and a low level of KRAS G12V. Healthy donor peripheral blood mononuclear cells can be transduced to express RAS TCR-1. Irrelevant TCR transduced T cells can be used as a negative transduced T cell control. TCR transduced T cells can be co-cultured with SNG-M tumor cells at an effector to target cell ratio of 3: 1. In some embodiments, the tumor cell line, Pane 03.27, can endogenously express HLA-C01:02 and KRAS G12V mutation. Healthy donor peripheral blood mononuclear cells can be transduced to express either RAS TCR-2 or RAS TCR-3. Non transduced T cells can be used as a negative control. TCR transduced T cells can be co-cultured with Pane 03.27 tumor cells at two different effector to target cell ratios (6: 1 and 12: 1). Pharmaceutical Compositions

[0351] The present disclosure provides pharmaceutical compositions comprising the cells manufactured by the methods described herein (e.g., Insta TCR process).

[0352] The immune cells described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g.. for use in treating or preventing a disease involving an antigen such as a cancer disease or an infectious disease.

[0353] The pharmaceutical composition can be a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

[0354] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. In some embodiments, the pharmaceutical composition does not comprise an adjuvant. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions ( e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune- stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, Interferon-a (INFa), INF-7, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or Lymphotoxin-a (LT-a). Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as MONTANIDE® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0355] The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., in a frozen or lyophilized/freeze-dried form) or in a "ready-to-use form" (i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form( e.g., a frozen pharmaceutical composition has to be thawed, or a freeze-dried pharmaceutical composition has to be reconstituted, e.g., by using a suitable solvent (e.g., deionized water, such as water for injection) or liquid (e.g., an aqueous solution)).

[0356] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0357] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0358] The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In some embodiments relating to the treatment of a particular disease, the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition, or symptoms thereof An effective amount of the pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0359] The pharmaceutical compositions of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.

[0360] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0361] The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants. [0362] The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

[0363] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0364] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

Use of the Engineered Immune Cells for Making a Medicament

[0365] Provided herein are uses of engineered immune cells described herein in the manufacture of a medicament. The medicament can be a population of immune cells expressing TCRs described herein. The population of immune cells (e.g., T cells, such as autologous T cells or allogeneic T cells) expressing the TCRs described herein can be formulated as a pharmaceutical composition with additional adjuvants or pharmaceutically acceptable carriers or excipients.

[0366] A pharmaceutical composition comprising an active agent such as an immune cell comprising TCRs described herein, in combination with one or more adjuvants can be formulated in conventional manner using one or more physiologically acceptable carriers, comprising excipients, diluents, and/or auxiliaries, e.g. , which facilitate processing of the active agents into preparations that can be administered. Proper formulation can depend at least in part upon the route of administration chosen. The agent(s) described herein can be delivered to a patient using a number of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular applications, as well as by inhalation. The active agents can be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. [0367] In some embodiments, a pharmaceutical composition comprised of the engineered immune cells can further comprise an acceptable additive in order to improve the stability of immune cells in the composition. Acceptable additives may not alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable carriers and/or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. Addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0368] When the compositions of engineered immune cells are considered for use in medicaments or any of the methods provided herein, it is contemplated that the composition can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are well understood to one or ordinary skill of the art and can be accomplished using commercially available kits.

[0369] Acceptable carriers can contain a compound that acts as a stabilizing agent, increases or delays absorption, or increases or delays clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion the compound can be complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13: 1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48: 119 135; and U.S. Pat. No. 5,391,377). [0370] For injectable formulations, the vehicle can be chosen from those known in art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. The formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co-glycolic)acid. These materials can be made into micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agent in injection grade water, liposomes and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.

[0371] In some instances, pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0372] In some embodiments, the engineered immune cells, particles, complexes or pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly, intratumorally, or peritumorally. In some embodiments, the immune effector cells, particles, complexes or pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the immune effector cells, particles, complexes or pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the immune effector cells, particles, complexes or pharmaceutical compositions are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the immune effector cells, particles, complexes or pharmaceutical compositions are formulated for intramuscular administration.

[0373] When administration is by injection, the active agent can be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer’s solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In another embodiment, the pharmaceutical composition does not comprise an adjuvant or any other substance added to enhance the immune response.

[0374] In addition to the formulations described previously, the active agents can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or use of a transdermal patch. Thus, for example, the agents can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0375] In some embodiments, a pharmaceutical composition can comprise the population of engineered immune cells produced according to any of the methods disclosed herein. In some embodiments, a pharmaceutical composition can comprise the engineered immune cells disclosed herein. In some embodiments, a pharmaceutical composition can comprise the population of engineered immune cells disclosed herein.

[0376] In some embodiments, any of the pharmaceutical compositions disclosed herein can be administered to a subject. In some embodiments, any of the pharmaceutical compositions disclosed herein can be administered to a subject to treat a disease or condition in a subject in need thereof. In some embodiments, the subject can be the same subject from which the biological sample is obtained.

[0377] In some embodiments, any population of engineered immune cells disclosed herein can be used in the manufacture of a medicament for treating a cancer in a subject. In some embodiments, any engineered immune cells disclosed herein can be used in the manufacture of a medicament for treating a cancer in a subject.

[0378] In some embodiments, the disease or condition that can be treated with the methods disclosed herein is abnormal growth of cells. In some embodiments, the disease or condition that can be treated with the methods disclosed herein is cancer. In some embodiments, the cancer is a malignant cancer. In some embodiments, the cancer is a benign cancer. In some embodiments, the cancer is an invasive cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid cancer.

[0379] The methods of the disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers to be treated by the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0380] Additionally, the disease or condition provided herein includes refractory or recurrent malignancies whose growth may be inhibited using the methods of treatment of the present disclosure. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non- small cell lung cancer, squamous cell carcinoma of the lung, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, a cancer to be treated by the methods of the present disclosure include, for example, carcinoma, squamous carcinoma (for example, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary). In some embodiments, a cancer to be treated by the methods of the present disclosure further include sarcomata (for example, myogenic sarcoma), leukosis, neuroma, melanoma, and lymphoma. In some embodiments, a cancer to be treated by the methods of the present disclosure is breast cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is triple negative breast cancer (TNBC). In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is ovarian cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is colorectal cancer. [0381] In some embodiments, a patient or population of patients to be treated with a pharmaceutical composition of the present disclosure have a solid tumor. In some embodiments, a solid tumor is a melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma. In some embodiments, a patient or population of patients to be treated with a pharmaceutical composition of the present disclosure have a hematological cancer. In some embodiments, the patient has a hematological cancer such as Diffuse large B cell lymphoma (“DLBCL”), Hodgkin’s lymphoma (“HL”), Non-Hodgkin’s lymphoma (“NHL”), Follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), or Multiple myeloma (“MM”). In some embodiments, a patient or population of patients to be treated having the cancer selected from the group consisting of ovarian cancer, lung cancer and melanoma.

[0382] Specific examples of cancers that can be prevented and/or treated in accordance with present disclosure include, but are not limited to, the following: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom’s macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma- induced bone osteosarcoma, Paget’s disease of bone, osteosarcoma, chondrosarcoma, Ewing’s sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget’s disease (including juvenile Paget’s disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing’s disease, prolactin- secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget’s disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, RAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as RAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, largecell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter); renal carcinoma; Wilms’ tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endothelio sarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinomas.

Gene Delivery

[0383] The nucleic acid molecules described herein can be delivered into the immune cells by various methods.

[0384] Gene delivery includes but is not limited to biological, chemical, mechanical, and physical methods. These include, for example, electroporation, lipid-based transfection, polymer-based transfections, or viral-based transfection.

[0385] Examples of biological methods include the use of viral vectors. Chemical based methods can use natural or synthetic compounds to form particles that facilitate the transfer of genes into cells. For example, calcium phosphate has been used to bind DNA and expose it to cultured cells to be integrated into the genome. Examples of mechanical methods may comprise microinjection, which may involve penetrating the cell membrane with the help of a micropipette in order to deliver nucleic acids to the cytoplasm. Physical methods may use physical force to perforate the cell membrane and introduce exogenous nucleic acids into the cells. Physical methods may be less invasive and require less cell contact. Physical method may include but are not limited electroporation, magnetoporation, sonoporation, or optoporation. In some cases, physical methods may comprise of electroporation.

[0386] The presentation disclosure may comprise the process of electroporation so as to induce gene delivery. Electroporation may comprise short-high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. The process may yield a high frequency of both stable transformation and transient gene expression. For example, with mammalian cells, cells are placed in suspension in an appropriate electroporation buffer and put into an electroporation cuvette. DNA may be added, and the cuvette may be connected to a power supply. The cells may then be subject to a high- voltage electrical pulse of defined magnitude and length. The cells may then be able to recover briefly before they placed in normal cell growth medium.

Particles

[0387] The present disclosure provides particles for delivering the nucleic acid sequences or molecules into the immune cells. In some embodiments, the particles may comprise further nucleic acids. In some embodiments, the particles comprise a third nucleic acid, or more than one third nucleic acid. In some embodiments, the particles contain a third and a fourth nucleic acid, or more than one third and/or fourth nucleic acid. In some embodiments, the particles contain a third and a fifth nucleic acid, or more than one third and/or fifth nucleic acid. In some embodiments, the particles contain a third, a fourth and a fifth nucleic acid, or more than one third, fourth and/or fifth nucleic acid.

[0388] To overcome the barriers to safe and effective nucleic acid delivery, nucleic acids may be administered with one or more delivery vehicles that protect the nucleic acids from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Such nucleic acid delivery vehicles may complex or encapsulate nucleic acids and include a range of materials, including polymers and lipids. In some embodiments, such nucleic acid delivery vehicles may form particles with nucleic acids, preferably DNA and/or RNA.

[0389] DNA or RNA, in particular mRNA, described herein may be present in particles comprising (i) the DNA and/or RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the DNA and/or RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged DNA and/or RNA are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid, in particular DNA and/or RNA, particles.

[0390] Different types of nucleic acid containing particles have been described previously to be suitable for delivery of DNA and/or RNA in particulate form (cf., e.g., Kaczmarek, J. C. el al., 2017, Genome Medicine 9, 60). For non-viral DNA and/or RNA delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acids from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0391] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0392] A "DNA particle", "RNA particle" or "DNA and RNA particle" can be used to deliver DNA and/or RNA to a target site of interest (e.g., cell, tissue, organ, and the like). A DNA and/or RNA particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid combines together with the nucleic acids to form aggregates, and this aggregation results in colloidally stable particles.

[0393] DNA and/or RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0394] A lipoplex (LPX) described herein is obtainable from mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes.

[0395] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

[0396] In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of DNA and/or RNA are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

[0397] In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with nucleic acids. In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact DNA and/or RNA — lipoplexes.

[0398] In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and DNA and/or RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged RNA (especially mRNA) results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and optionally additional lipids, such as DOPE or DSPC.

[0399] In general, a lipid nanoparticle (LNP) is typically obtainable from direct mixing of DNA and/or RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0400] In some embodiments, LNPs comprise or consist of a cationic/cationically ionizable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the DNA and/or RNA LNPs described herein the DNA and/or RNA (in particular, mRNA) is bound by cationically ionizable lipid that occupies the central core of the LNP. In some embodiments, polymer- conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.

[0401] In some embodiments, DNA and/or RNA (e.g., mRNA) described herein may be noncovalently associated with a particle as described herein. In embodiments, the DNA and/or RNA (especially mRNA) may be adhered to the outer surface of the particle (surface RNA (especially surface mRNA)) and/or may be contained in the particle (encapsulated DNA and/or RNA (especially encapsulated mRNA)). [0402] In some embodiments, the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0403] In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, from about 200 nm to about 250 nm, or from about 80 to about 150 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 tun to about 160 nm, from about 80 run to about 150 nm or from about 80 nm to about 120 nm.

[0404] DNA and/or RNA particles (especially mRNA particles) described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the DNA and/or RNA particles can exhibit a poly dispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0405] The N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations may be formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles can be favorable for transfection. In that case, DNA and/or RNA is considered to be completely bound to nanoparticles.

[0406] The present disclosure describes compositions comprising DNA and/or RNA (especially mRNA) and at least one cationic or cationically ionizable lipid which associates with the DNA and/or RNA to form DNA and/or RNA particles and formulations comprising such particles. The DNA and/or RNA particles may comprise DNA and/or RNA which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, they are not able to virally infect cells.

[0407] Suitable cationic or cationically ionizable lipids are those that form DNA and/or RNA particles and are included by the term "particle forming components" or "particle forming agents". The term "particle forming components" or "particle forming agents" relates to any components which associate with DNA and/or RNA to form DNA and/or RNA particles. Such components include any component which can be part of DNA and/or RNA particles. [0408] In some embodiments, DNA and/or RNA particles (especially mRNA particles) comprise more than one type of DNA and/or RNA molecules, where the molecular parameters of the DNA and/or RNA molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[0409] In particulate formulation, it is possible that each DNA and/or RNA species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one DNA and/or RNA species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each DNA and/or RNA species separately (typically each in the form of an DNA and/or RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific DNA and/or RNA species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of DNA and/or RNA-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that all DNA and/or RNA species of the pharmaceutical composition are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all DNA and/or RNA species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one DNA and/or RNA species. In a combined particulate composition different DNA and/or RNA species are typically present together in a single particle.

Polymers

[0410] Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense the negatively charged DNA and/or RNA into particles, in particular nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly([P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0411] A "polymer," as used herein, is given its ordinary meaning, z.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, z.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

[0412] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, z.e., comprising one or more regions each comprising a first repeat unit (.e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0413] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.

[0414] In certain embodiments, polymer may be protamine or poly alkyleneimine.

[0415] The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0416] According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0417] In one embodiment, the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75.102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

[0418] Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

[0419] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In one embodiment, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0420] Particles described herein may also comprise polymers other than cationic polymers, i.e. , non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

Lipids

[0421] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0422] As used herein, the term "hydrophobic" refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (.e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution. [0423] The term "hydrocarbon" includes non-cyclic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0424] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety which may have one to thirty, typically one to twenty, often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0425] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be six to thirty, typically six to twenty often six to eighteen. Generally, the maximal number of carboncarbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0426] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), 1, 2, 3, 4, 5, or

6, more preferably 1 or 2 carbon-carbon triple bonds.

[0427] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety which may have one to thirty, typically two to twenty, often four to twelve carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octadecmethylene, and the like.

[0428] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve. Generally, the maximal number of carboncarbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0429] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).

[0430] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. [0431] The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2- dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).

[0432] As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0433] The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances, in particular amphiphilic substances, that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function- spacer- lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

[0434] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, sterol-containing metabolites such as cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0435] Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more cis double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

[0436] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0437] The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0438] Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N- acyl- sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glyco sphingolipids such as cerebrosides and gangliosides.

[0439] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0440] Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3- deoxy-D-manno-octulosonic acid (Kdo) residues.

[0441] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0442] According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH. Cationic/Cationically ionizable lipids

[0443] In some embodiments, the DNA and/or RNA compositions and formulations and nucleic acid particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid-like materials) which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0444] As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

[0445] In some embodiments, a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0446] As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.

[0447] In some embodiments, the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.

[0448] Examples of cationic or cationically ionizable lipids include, but are not limited to N,N- dimethy 1-2, 3 -dioley loxypropylamine (DODMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP); l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1 ,2-di oleo yl-3-dimethylammonium-propane (DODAP); 1 ,2-diac yloxy-3 -dimethylammonium propanes; l,2-dialkyloxy-3- dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), 1,2-dimyristoy 1-3 -trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3- dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9, 1 2-oc- tadecadienoxy)propane (CLinDMA), 2-[5 '-(cholest-5-en-3-beta-oxy)-3 '-oxapentoxy)-3- dimethyl-l-(ci s,ci s-9', 1 2'-octadecadienoxy)propane (CpLinDMA), N, N-dimethy 1-3,4- dioleyloxybenzylamine (DMOB A), 1 ,2- N, N ¹ -dioley Icarbamy 1 -3 -dimethylaminopropane

(DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoley Icarbamy 1 -3 -dimethylaminopropane (DLincarbD AP) , 1 ,2-Dilinoleoylcarbamy 1-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[ 1 ,3 ] -dioxolane (DLin-K-XTC2- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[ 1 ,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 1-tetraen-l 9-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N- (2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide

(DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(ci s-9-tetradecenyloxy)-l-prop anaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-prop anaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2- Aminoethyl)-N ,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide ((PAE- DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium

(DOBAQ), 2-( {8-[(3 (P)-cholest-5-en-3-yloxy]octyl 1 oxy)-N ,N-dimethyl-3-[(9 Z,1 2Z)- octadeca-9, 1 2-di en-l-yloxy]prop an-l-amine (Octyl-CLinDMA), 1 ,2-dimyristoyl-3- dimethyl ammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Ni-[2-(( 1 S)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl] -3, 4-di[oleyloxy] -benzamide (MVLS), 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethypthio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan- 1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecy 1-3-02- dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)- [2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N 12-5), l-[2-[bis(2-hydroxydodecyl)amino] ethyl- [2- [4- [2- [bis(2 hydroxydodecyDamino]ethyllpiperazin-l-yl] ethyl] amino] dodecan-2-ol (lipidoid C 12-200). [0449] In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

[0450] DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows:

[0451] DODMA is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows:

[0452] In some embodiments, the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.

Additional lipids

[0453] The DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), noncationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. Optimizing the formulation of DNA and/or RNA particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of nucleic acid delivery. [0454] One or more additional lipids may or may not affect the overall charge of the DNA and/or RNA particles. In some embodiments, the one or more additional lipids are a noncationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. [0455] In some embodiments, the DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.

[0456] Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important DNA and/or RNA particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the DNA and/or RNA. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1:9, about 4: 1 to about 1:2, about 4: 1 to about 1: 1, about 3: 1 to about 1: 1, or about 3: 1 to about 2: 1.

[0457] In some embodiments, the one or more additional lipids comprised in the DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.

[0458] In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacy Ipho sphatidy Icholines , such as distearoylphosphatidylcholine (DSPC), dioleoy Ipho sphatidy Icholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoy Ipho sphatidy Icholine (D APC ) , dibehenoy Ipho sphatidy Icholine (DBPC), ditrico s anoy Ipho sphatidy Icholine (DTPC) , dilignoceroylphatidy Icholine (DLPC), palmitoyloleoyl-phosphatidylcholine : (POPC), 1,2-di-O-octadecenyl-sn-gl ycero-3- phosphocholine (18 :0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3 - phosphocholine (OChemsPC), 1 -hexadecyl- sn- glycero-3 -phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl- phosphatidylethanolamine (DP yPE), 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3 -phospho-(l '-rac-glycerol) (DPPG), 1 - palmitoyl -2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), N-p almitoyl-D- erythro- sphingosylphosphorylcholine (SM), and further phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0459] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0460] Thus, in some embodiments, the DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

[0461] In some embodiments, the DNA and/or RNA particles (especially the particles comprising mRNA) described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.

[0462] DSPC is a neutral phospholipid. The structure of DSPC may be represented as follows: [0463] DOPE is a neutral phospholipid. The structure of DOPE may be represented as follows:

The structure of cholesterol may be represented as follows:

[0464] In some embodiments, DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein do not include a polymer conjugated lipid such as a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0465] In some embodiments, the additional lipid (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle. In some embodiments, the additional lipid (e.g., one or more phospholipids and/or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.

[0466] In some embodiments, the additional lipid comprises a mixture of: (i) a phospholipid such as DOPE; and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of the phospholipid such as DOPE to the cholesterol or a derivative thereof is from about 9:0 to about 1: 10, about 2: 1 to about 1:4, about 1: 1 to about 1:4, or about 1: 1 to about 1:3.

Polymer-conjugated lipids

[0467] In some embodiments, DNA and/or RNA compositions and formulations and DNA and/or RNA particles described herein may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto. In some embodiments, a polymer-conjugated lipid is a PEG- conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art. In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine portion.

[0468] In some embodiments, a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a polymer-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

Polyethylene glycol (PEG)-conjugated lipids

[0469] In some embodiments, DNA and/or RNA compositions/formulations and DNA and/or RNA particles described herein comprise a PEG-conjugated lipid.

[0470] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R12 and R13 is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. [0471] In some embodiments of this formula, each of R12 and R13 is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms. In some embodiments of this formula, R12 and R13 are identical. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 16 carbon atoms. In some embodiments of this formula, R12 and R13 are different. In some embodiments, one of R12 and R13 is a straight alkyl chain containing 12 carbon atoms and the other of R12 and R13 is a straight alkyl chain containing 14 carbon atoms. [0472] In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45. In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

[0473] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as 1 -(monomethoxy-polyethylene glycol)-2,3- dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propy l-l-O- (co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as to-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(w methoxy(polyethoxy)ethyl)carbamate, and the like. In some embodiments, the PEG- conjugated lipid (pegylated lipid) is or comprises 2- [(polyethylene glycol)-2000]-N,N- ditetradecylacetamide. In some embodiments, the pegylated lipid has the following structure:

[0474] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is DMG-PEG 2000, e.g., having the following structure:

[0475] In some embodiments, the PEG-conjugated lipid (pegylated lipid) has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N- [(e)-methoxy poly (ethylene glycol)2000)carbamoy 1 ] - 1 ,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000).

[0476] In some embodiments, DNA and/or RNA compositions/formulations described herein may comprise one or more PEG-conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0477] In some embodiments, the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the DNA and/or RNA compositions/formulations and DNA and/or RNA particles described herein.

Embodiments of Lipoplex Particles

[0478] In some embodiments of the present disclosure, the DNA and/or RNA described herein may be present in DNA and/or RNA lipoplex particles.

[0479] Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic nucleic acids. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact DNA and/or RNA — lipoplexes.

[0480] In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0481] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3: 1 to about 1: 1. In specific embodiments, the molar ratio may be about 3: 1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1: 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.

[0482] DNA and/or RNA lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the DNA and/or RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the DNA and/or RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the DNA and/or RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the DNA and/or RNA lipoplex particles have an average diameter of about 400 nm.

[0483] The DNA and/or RNA lipoplex particles and compositions comprising DNA and/or RNA lipoplex particles described herein are useful for delivery of DNA and/or RNA to a target tissue after parenteral administration, in particular after intravenous administration.

[0484] Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen- presenting cells, in particular dendritic cells. Accordingly, following administration of the DNA and/or RNA lipoplex particles, DNA and/or RNA accumulation and/or DNA and/or RNA expression in the spleen occurs. Thus, DNA and/or RNA lipoplex particles of the disclosure may be used for expressing DNA and/or RNA in the spleen. In an embodiment, after administration of the DNA and/or RNA lipoplex particles, no or essentially no DNA and/or RNA accumulation and/or DNA and/or RNA expression in the lung and/or liver occurs. In some embodiments, after administration of the DNA and/or RNA lipoplex particles, DNA and/or RNA accumulation and/or DNA and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, DNA and/or RNA lipoplex particles of the disclosure may be used for targeting DNA and/or RNA, e.g., DNA and/or RNA encoding an antigen or at least one epitope, to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular preferred if the DNA and/or RNA administered is DNA and/or RNA encoding vaccine antigen. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen.

[0485] The electric charge of the DNA and/or RNA lipoplex particles of the present disclosure is the sum of the electric charges present in the at least one cationic lipid and the electric charges present in the DNA and/or RNA. The charge ratio is the ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the DNA and/or RNA. The charge ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the DNA and/or RNA is calculated by the following equation: charge ratio=[(cationic lipid concentration (mol)) * (the total number of positive charges in the cationic lipid)] / [(DNA and/or RNA concentration (mol)) * (the total number of negative charges in DNA and/or RNA)]. The concentration of DNA and/or RNA and the at least one cationic lipid amount can be determined using routine methods by one skilled in the art.

[0486] In some embodiments, at physiological pH the charge ratio of positive charges to negative charges in the DNA and/or RNA lipoplex particles is from about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the charge ratio of positive charges to negative charges in the DNA and/or RNA lipoplex particles at physiological pH is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0. Embodiments of Lipid nanoparticles (LNPs)

[0487] In some embodiments, DNA and/or RNA described herein is present in the form of lipid nanoparticles (LNPs). LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer- conjugated lipid such as PEG-lipid. LNPs may be prepared by mixing lipids dissolved in ethanol with DNA and/or RNA in an aqueous buffer.

[0488] In some embodiments, in the DNA and/or RNA LNPs described herein the DNA and/or RNA is bound by cationically ionizable lipid that occupies the central core of the LNP. Polymer-conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, cholesterol and cationically ionizable lipid can be distributed throughout the LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and polymer-conjugated lipids. In some embodiments, the LNP comprises a cationically ionizable lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and the DNA and/or RNA, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the LNP comprises from 35 to 65 mol percent, 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid. In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid. In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the DNA and/or RNA, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.

[0489] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.

[0490] In some embodiments, the cationically ionizable lipid component of the LNPs has the structure of Formula (III): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of LI or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of LI or L2 is — O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cyclo alkenylene; Ra is H or Cl -C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, ORS, CN, -C(=O)OR4, -OC(=O)R4 or — NR5C(=O)R4;R4 is Cl -C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1 or 2. [0491] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

(ISA) ( BIB! wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R6 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0492] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0493] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID): , wherein y and z are each independently integers ranging from 1 to 12.

[0494] In any of the foregoing embodiments of Formula (III), one of LI or L2 is -O(C=O)-. For example, in some embodiments each of LI and L2 are -O(C=O)-. In some different embodiments of any of the foregoing, LI and L2 are each independently -(C=O)O- or -0(C=0)- . For example, in some embodiments each of LI and L2 is -(C=O)O-.

[0495] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): [0496] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ) :

(II II) (110)

[0497] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0498] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0499] In some of the foregoing embodiments of Formula (III), R6 is H. In other of the foregoing embodiments, R6 is C1-C24 alkyl. In other embodiments, R6 is OH.

[0500] In some embodiments of Formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is linear C1-C24 alkylene or linear Cl- C24 alkenylene.

[0501] In some other foregoing embodiments of Formula (III), R1 or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1 and R2 each, independently have the following structure: wherein: R7a and R7b are, at each occurrence, independently H or Cl-C 12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b and a are each selected such that R1 and R2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0502] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl. In different embodiments of Formula (III), R1 or R2, or both, has one of the following structures:

[0503] In some of the foregoing embodiments of Formula (III), R3 is OH, CN, -C(=O)OR4, - OC(=O)R4 or — NHC(=O)R4. In some embodiments, R4 is methyl or ethyl.

[0504] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below.

[0505] Representative Compounds of Formula (III).

[0506] Further representative cationically ionizable lipids are as follows: [0507] In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a polymer conjugated lipid.

[0508] In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer conjugated lipid is a pegylated lipid, e.g., DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0509] In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0510] In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and a pegylated lipid.

[0511] In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and a pegylated lipid. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and a pegylated lipid.

[0512] In some embodiments, the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC- 0366, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and PEG2000-C- DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159. In some embodiments, DNA and/or RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159.

[0513] 3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l l-yl 5- (dimethylamino)pentanoate

[0515] ALC-0315: ((4-hydroxybutypazanediy l)bis(hexane-6,l-diyObis(2-hexyldecanoate) /

6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate

[0516] DMG-PEG 2000:

[0517] PEG2000-C-DMA: 3-N-[(o)-Methoxy poly(ethylene glycol)2000) carbamoyl]-l,2- dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA or Methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000)) wherein n has a mean value ranging from 30 to 60, such as about 50.

[0518] ALC-0159: 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2-[2-(o- methoxy (polyethyleneglyco 12000) ethoxy] -N,N-ditetradecylacetamide

[0519] DSPC: l,2-Distearoyl-sn-glycero-3 -phosphocholine

[0521] The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6. [0522] The particles described herein may comprise a hydrophobic moiety (e.g., lipid) having a binding moiety covalently attached thereto. This hydrophobic moiety having a binding moiety covalently attached thereto is also referred to herein as "connector compound". The hydrophobic moiety of the connector compound relates to the part of the connector compound that integrates into the particle comprising a payload. The binding moiety of the connector compound relates to the part of the connector compound that forms the binding partner for the docking compound. Generally, the connector compound is non-covalently incorporated into the particle comprising a pay load, i.e., it forms an integral part of the particle, and the binding moiety of the connector compound is covalently attached to a hydrophobic moiety in a manner such that it is available for binding to the docking compound.

[0523] In some embodiments, the binding moiety of the connector compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).

[0524] In some embodiments, the binding moiety of the connector compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag) and is chemically linked, e.g., through a linker, to the hydrophobic moiety (e.g., lipid).

[0525] The connector compound used herein comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle. In some embodiments, the hydrophobic component comprises a moiety selected from vitamin E, dialkylamine, e.g., dimyristylamine (DMA), diacylglyceride, e.g., 1,2-dimyristoyl-sn-glycerol (DMG) and ceramide. In some embodiments, the hydrophobic moiety comprises two C8-C24 hydrocarbon chains. In some embodiments, the hydrophobic moiety comprises two Cl 0-C18 hydrocarbon chains.

[0526] In some embodiments, the connector compound used herein has as a hydrophobic group (e.g., lipid) a phospholipid, e.g., a biodegradable phospholipid such as phosphatidylethanolamine. In some embodiments, the connector compound used herein has as a hydrophobic group (e.g., lipid) a glycerophospholipid. In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylpho sphatidyl ethanol amine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE

(dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, as a phospholipid, DSPE will be used for its qualities of stability in the particles described herein. Moreover, as hydrophobic group (e.g., lipid), a compound having at least one alkyl chain providing hydrophobic anchoring to a particle as described herein may be used.

[0527] In some embodiments, the connector compound comprises a polymer. In some embodiments, the hydrophobic moiety (e.g., lipid) of the connector compound and the binding moiety of the connector compound are connected through the polymer.

[0528] In some embodiments, the polymer is a hydrophilic polymer and the connector compound comprises an amphiphilic derivative of the polymer. In some embodiments, the amphiphilic derivative of a polymer comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle and a hydrophilic component of the polymer facing the outside of said particle, conferring hydrophilic properties at the surface thereof. In some embodiments, the amphiphilic derivatives of a polymer is inserted into the particle via its hydrophobic end. Consequently, the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle. In some embodiments, the polymer portion of the amphiphilic derivative contributes to conferring stealth properties on the particles. In some embodiments, the plasmatic half-life of the particles described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the particles to accumulate at the target cells and to liberate therein their contents (pay load) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

[0529] The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and/or degraded, or to be hardly detected and then sequestered and/or degraded, and/or to be detected and then sequestered and/or degraded late, by the immune system of the host to which they are administered.

[0530] Macrophages constitute one of the most important components of the immune system and play a predominant role in eliminating foreign particles, including liposomes and other colloidal particles, from the blood circulation. At the molecular level, the clearance of particles takes place in two steps: opsonization by the depositing of serum proteins (or "opsonins") at the surface of the particles followed by recognition and capture of the opsonized particles by macrophages.

[0531] Modification of the surface of particles with chains of hydrophilic and flexible polymers, e.g., polymers of the poly(ethylene glycol) type, confers them a steric protection by preventing the opsonins reaching the surface of the particles.

[0532] In some embodiments, the amphiphilic derivative of a polymer used herein has as a hydrophobic group (e.g., lipid) as specified herein. In some embodiments, the amphiphilic derivative of a polymer used herein has as a hydrophobic group (e.g., lipid) a phospholipid, e.g., a biodegradable phospholipid such as phosphatidylethanolamine. In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, as a phospholipid, DSPE will be used for its qualities of stability in the particles described herein. Moreover, as hydrophobic group (e.g., lipid), a compound having at least one alkyl chain providing hydrophobic anchoring to a particle as described herein may be used. [0533] In some embodiments, the polymer for use herein is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (PDX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).

[0534] In some embodiments, a polymer is designed to sterically stabilize a particle by forming a protective hydrophilic layer. In some embodiments, a polymer can reduce association of a particle with serum proteins and/or the resulting uptake by the reticuloendothelial system when such particles are administered in vivo.

[0535] In some embodiments, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.

[0536] In some embodiments, the PEG moiety of the amphiphilic derivative of a polymer has a molecular weight of 1000 or more. In some embodiments, the PEG moiety of the amphiphilic derivative of a polymer comprises 10 units or more of formula (O-CH2-CH2)n. In some embodiments, the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0537] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.

[0538] In some embodiments, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as the amphiphilic derivative of a polymer.

[0539] In some embodiments, a pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.

[0540] In some embodiments, a pSar comprises the structure of the following general formula: , wherein s is the number of sarcosine units.

[0541] In some embodiments, the PDX and/or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 PDX and/or POZ repeating units.

[0542] In some embodiments, the PDX and/or POZ polymer comprises the following general formula: wherein a is an integer between 1 and 2; Rl l is alkyl, in particular Cl-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of PDX and/or POZ repeating units.

[0543] In some embodiments, the PDX and/or POZ polymer is a polymer of PDX and comprises repeating units of the following general formula: . In some embodiments, the PDX and/or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:

[0544] In any of the above embodiments of formulas, m (z.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70. In certain embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0545] In some embodiments, the PDX and/or POZ polymer is a copolymer comprising repeating units of the following general formulas: wherein the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.

[0546] In some embodiments of the oxazolinylated and/or oxazinylated hydrophobic moiety (e.g., lipid), the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50. [0547] In some of the above embodiments, R11 at each occurrence (z.e., in each repeating unit) may be the same alkyl group (e.g., Rl l may be methyl in each repeating unit). In some alternative embodiments, Rl l in at least one repeating unit differs from Rl l in another repeating unit (e.g., for at least one repeating unit R11 is one specific alkyl (such as ethyl), and for at least one different repeating unit Rl l is a different specific alkyl (such as methyl)). For example, each R11 may be selected from two different alkyl groups (such as methyl and ethyl) and not all R11 are the same alkyl.

[0548] In any of the above embodiments, Rll preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rl l is methyl or each Rl l is ethyl. In some alternative embodiments, Rl l is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit R11 is methyl, and in at least one repeating unit Rl l is ethyl.

[0549] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.

[0550] In some embodiments, the polymer comprises the following general formula:

, wherein X2 and XI taken together are optionally substituted amide, optionally substituted thioamide or ester; Y is -CH2-, -(CH2)2-, or -(CH2)3- ; z is 2 to 24; and n is 1 to 100. In some embodiments, (i) when XI is -C(O)- then X2 is -NR1-

; (ii) when XI is -NR1- then X2 is -C(O)-; (iii) when XI is -C(S)- then X2 is -NR1-; (iv) when XI is -NR1- then X2 is -C(S)-; (v) when XI is -C(O)- then X2 is -O-; or (vi) when XI is -O- then X2 is -C(O)-; wherein R1 is hydrogen or Cl-8 alkyl. In some embodiments, XI is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or Cl-8 alkyl. In some embodiments, XI is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or methyl. In some embodiments, XI is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen. In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-. In some embodiments, Y is -(CH2)2-.

[0551] In some embodiments, the polymer comprises the following general formula:

, wherein R1 is hydrogen or Cl-8 alkyl; z is 2 to 24; and n is 1 to 100. In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0552] In some embodiments, the polymer comprises the following general formula:

, wherein R1 is hydrogen or Cl-8 alkyl; and n is 1 to 100.

In some embodiments of the above formulas, R1 is hydrogen or methyl. In some embodiments, R1 is hydrogen.

[0553] In some embodiments, the polymer comprises the following general formula: wherein n is 1 to 100. In some embodiments of the above formulas, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0554] In some embodiments, the molar proportion of the amphiphilic derivative of a polymer integrated into the particles is between 0.5 and 20 mol% of the lipid molecules making up the particle, preferably between 1 and 10 mol%.

[0555] In some embodiments, the connector compound comprises the following general formula:

L-X1-P-X2-B, wherein P comprises a polymer; L comprises a hydrophobic moiety e.g., lipid) 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; and X2 is absent or a second linking moiety. In some embodiments, XI comprises a carbonyl group. In some embodiments, L comprises a phosphatidylethanolamine which may be linked to P by an amide group. In some embodiments, X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a thiol or cysteine group of a compound comprising the binding moiety. [0556] In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group.

[0557] In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer which provides stealth property, extends circulation halflife and/or reduces non-specific protein binding or cell adhesion. In some embodiments, P comprises a polymer selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (PDX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments, P comprises polyethylene glycol (PEG); e.g., PEG as described above.

[0558] In some embodiments, L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethylene glycol-conjugate (D SP E-PEG).

[0559] In some embodiments, the connector compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the binding moiety.

[0560] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0561] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the binding moiety comprises the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.

[0562] In some embodiments, the connector compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.

[0563] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula:

L-X1-P-X2-B wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety. [0564] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein B comprises the binding moiety.

[0565] In some embodiments of the above formulas, B comprises a moiety comprising the structure - N-peptide-C(O)-NH2.

[0566] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein P, X2 and B are as described above and R1 and R2 independently comprise an alkyl moiety. In some embodiments, at least one, e.g., each alkyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each alkyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of alkyl moieties include -(CH2)17CH3 (stearyl), -(CH2)15CH3 (palmityl), and -(CH2)13CH3 (myristyl). In some embodiments, R1R2N- in the above formula is 1,2-dimyristylamine, wherein both alkyl groups are - (CH2)13CH3 (myristyl).

[0567] In some embodiments, the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 14. In some embodiments, R1 and R2 in the above formula are -(CH2)13CH3 (myristyl) and the polymer P comprises the following general formula: wherein n is 14.

[0568] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein P, X2 and B are as described above and each of Rtl and Rt2 is independently H or methyl. In some embodiments, Rtl and Rt2 are both methyl. In some embodiments, Rtl is methyl, and Rt2 is H. In some embodiments, Rtl is H, and Rt2 is methyl. In some embodiments, Rtl and Rt2 are both H.

[0569] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein P, X2 and B are as described above.

[0570] In some embodiments, the polymer P in the above formulas comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0571] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein XI, P, X2 and B are as described above and R1 and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2)16C(O)- (stearoyl), CH3(CH2)14C(O)- (palmitoyl), and CH3(CH2)12C(O)- (myristoyl). In some embodiments, both acyl groups are CH3(CH2)16C(O)- (stearoyl). In some embodiments, both acyl groups are CH3(CH2)12C(O)- (myristoyl). In some embodiments, XI is absent or comprises -HPO3- (CH2)n-NH-, wherein n is 1 to 5, e.g., 2.

[0572] In some embodiments, the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0573] In some embodiments, the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.

[0574] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula:

[0575] wherein P, X2 and B are as described above and R1 and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2)16C(O)- (stearoyl), CH3(CH2)14C(O)- (palmitoyl), and CH3(CH2)12C(O)- (myristoyl). In some embodiments, both acyl groups are CH3(CH2)16C(O)- (stearoyl). In some embodiments, both acyl groups are CH3 (CH2)12C(O)- (myristoyl).

[0576] In some embodiments, the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0577] In some embodiments, n is 8 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl). In some embodiments, n is 14 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl).

[0578] In some embodiments, n is 8 and RI and R2 are CH3(CH2)12C(O)- (myristoyl). In some embodiments, n is 14 and Rl and R2 are CH3(CH2)12C(O)- (myristoyl).

[0579] In some embodiments, the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.

[0580] In some embodiments, s is 20 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl). In some embodiments, s is 20 and RI and R2 are CH3(CH2)12C(O)- (myristoyl).

[0581] In some embodiments, X2 in the above formulas comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a compound comprising a thiol or cysteine group. In some embodiments, the compound comprising a thiol or cysteine group comprises the formula SH(CH2)nC(O)-, wherein n ranges from 1 to 5. In some embodiments, n is 2. In some embodiments, X2 comprises a thiosuccinimide moiety.

[0582] In some embodiments, X2 comprises the following general formula: In some embodiments, X2 comprises the following general formula:

, wherein nl and n2 are independently 1 to 5. In some embodiments, nl is 1 and n2 is 2. In some embodiments, nl is 2 and n2 is 1.

[0583] The present disclosure provides in one aspect, a connector compound as described herein. In some embodiments of the connector compound, the binding moiety comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0584] The present disclosure provides in one aspect, a compound having the following general formula: L-X 1 -P-X2-B, wherein P comprises a polymer; L comprises a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer; B comprises an epitope tag, e.g., an ALFA- tag such as an ALFA-tag described herein, 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, L comprises a phosphatidylethanolamine which may be linked to P by an amide group. In some embodiments, X2 comprises the reaction product of a thiol or cysteine reactive group, e.g. , a maleimide group, with a thiol or cysteine group of a compound comprising the epitope tag. In some embodiments, X2 comprises a thio succinimide moiety.

[0585] In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group. [0586] In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer which provides stealth property, extends circulation halflife and/or reduces non-specific protein binding or cell adhesion. In some embodiments, P comprises a polymer selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (PDX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments, P comprises polyethylene glycol (PEG); e.g., PEG as described above.

[0587] In some embodiments, L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0588] In some embodiments, the connector compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the epitope tag.

[0589] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0590] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the epitope tag comprises the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the epitope tag. In some embodiments, n is 2.

[0591] In some embodiments, the connector compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-{maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the epitope tag. In some embodiments, n is 2.

[0592] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: , wherein

B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0593] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: wherein X2 is as described above, R1 and R2 are CH3(CH2)16C(O)- (stearoyl) or CH3(CH2)12C(O)- (myristoyl), polymer P comprises the following general formula: , wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17, e.g., 8 or 14, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0594] In some embodiments, n is 8 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl). In some embodiments, n is 14 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl). In some embodiments, n is 8 and R1 and R2 are CH3(CH2)12C(O)- (myristoyl). In some embodiments, n is 14 and R1 and R2 are CH3(CH2)12C(O)- (myristoyl).

[0595] In some embodiments, X2 comprises the following general formula:

[0596] In some embodiments, the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula: , wherein X2 is as described above, R1 and

R2 are CH3(CH2)16C(O)- (stearoyl) or CH3(CH2)12C(O)- (myristoyl), polymer P comprises the following general formula: , wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40, e.g., 20 or 23, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0597] In some embodiments, s is 20 and R1 and R2 are CH3(CH2)16C(O)- (stearoyl). In some embodiments, s is 20 and R1 and R2 are CH3(CH2)12C(O)- (myristoyl). In some embodiments, X2 comprises the following general formula:

[0598] In some embodiments, B comprises a moiety comprising the structure -N-peptide- C(O)-NH2, wherein peptide comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0599] The present disclosure provides in one aspect, a connector compound as described above which is integrated in a particle (e.g., a particle as described herein) via a hydrophobic component (e.g., lipid component) of the connector compound.

Vectors and Delivery of Nucleic Acids

[0600] Vectors containing the recombinant nucleic acids encoding TCRs described herein can be delivered to host cells for expression and processing.

[0601] Terms such as “transferring”, “introducing” or “transfecting” are used interchangeably herein and relate to the introduction of nucleic acids, in particular exogenous or heterologous nucleic acids, into a cell.

[0602] Cells can be transfected with any carriers with which nucleic acid can be associated, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated, resulting in increased stability of the nucleic acid compared to naked nucleic acid. Carriers useful according to the present disclosure include, for example, lipid-containing carriers such as cationic lipids, liposomes, in particular cationic liposomes, and micelles, and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used according to the present disclosure.

[0603] In various embodiments, a nucleic acid encoding a TCR disclosed herein is operably linked to a promoter. Furthermore, the present disclosure provides a vector, e.g., a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or particle and/or vector to be used in gene therapy, which comprises one or more of the nucleic acids as disclosed above. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid. The vector comprises the nucleic acid insert, which encodes the polypeptide or protein desired for expression in a cell, such as a host cell. For the purposes of the disclosure, an insert can be a nucleic acid encoding a TCR; an alpha chain or a beta chain or both of a TCR. The term “incorporating” a nucleic acid sequence in a vector can mean preparing a suitable expression vector with an insert comprising said nucleic acid sequence. An “expression vector” is a vector that can direct the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment. “Retroviruses” are viruses having an RNA genome. A vector that encodes a core virus is also known as a “viral vector.” There are a large number of available viral vectors that are suitable for use with the invention, including those identified for human gene therapy applications, such as those described by Pfeifer and Verma (Pfeifer, A. and I. M. Verma. 2001. Ann. Rev. Genomics Hum. Genet. 2: 177-211). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus -derived vectors, e.g., Moloney murine leukemia virus (MLV)- derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and maedi/visna virus. Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian target cells with viral particles containing TCRs transgenes are well known in the art and have been previous described, for example, in U.S. Pat. No. 8,119,772; Walchli et al., 2011, PLoS One 6:327930; Zhao etal., J. Immunol., 2005, 174:4415-4423; Engels et al., 2003, Hum. Gene Ther. 14: 1155-68; Frecha et al., 2010, Mol. Ther. 18: 1748-57; Verhoeyen et al., 2009, Methods Mol. Biol. 506:97-114. Retroviral and lentiviral vector constructs and expression systems are also commercially available. In some embodiments, a viral vector is used to introduce the non-endogenous nucleic acid sequence encoding TCRa chain specific for the peptide antigen into the hematopoietic progenitor cells. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selection markers, which can confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. Where the viral vector genome comprises more than one nucleic acid sequence to be expressed in the host cell as separate transcripts, the viral vector can also comprise additional sequence between the two (or more) transcripts allowing bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide. Other vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus -based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., 1998, Gene Ther. 5: 1517-30). Other vectors include those derived from baculoviruses and alpha-viruses. (Jolly D J. 1999. Emerging viral vectors, pp 209-40 in Friedmann T. ed. 1999. The development of human gene therapy. New York: Cold Spring Harbor Lab).

[0604] A vector can include nucleic acid sequences that permit the nucleic acid to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. A vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid.

[0605] In some embodiments, provided herein is a vector comprising a nucleic acid encoding a TCR disclosed herein. In some embodiments, the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, pox virus, alpha virus, vaccina virus, hepatitis B virus, human papillomavirus or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, a cationic lipid, a cationic polymer, a metallic nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cellpenetrating peptide, or a liposphere.

[0606] Presented herein are constructs, for example, nucleic acid constructs that encode an alpha chain and a beta chain of a TCR for expression in a cell. In some embodiments, the constructs comprise one or more polynucleotides encoding a TCR alpha chain and a TCR beta chain. In some embodiments, the polynucleotides are incorporated in a suitable vector. In some embodiments, the polynucleotides encoding the alpha chain and the beta chain are incorporated in the same vector. In some embodiments, the polynucleotides encoding the alpha chain and the beta chain are incorporated in different vectors, and both vectors are delivered for expression in the single cell.

[0607] In some embodiments, a cell (e.g., population of T cells described herein) can be transduced or transfected with a nucleic acid encoding a TCR, wherein the cell is capable of expressing the TCR and the cell is used as a therapeutic. In some embodiments the cell is derived from a subject or a host, wherein the subject or the host is a human. In some embodiments, the cell is a lymphocyte cell. In some embodiments, the T-lymphocyte. In some embodiments, the cell is a lymphocytic precursor cell. In some embodiments, the cell is a T lymphocyte precursor cell. In some embodiments, the cell is a T lymphocyte progenitor cell. In some embodiments the cell is a thymocyte.

[0608] In some embodiments, the T cells are immature T cells. In some embodiments, the T cells are antigen naive T cells. The host cell can be cultured ex vivo for 1, 2, 3, 4, 5 or more days for monitoring and recover after transfection or transduction with the polynucleotide(s) encoding the TCR.

EXAMPLES

Example 1. Enhanced PRAME Insta TCR Construct

[0609] Conventional engineered cell therapy manufacturing comprises polyclonal T cell activation followed by transduction with one or more transgene, typically retrovirus or lentivirus. This process can lead to high efficiency and large numbers of engineered cells but requires extensive time in cell culture. To overcome these challenges, a transposon-based platforms was developed, in which transgenes are introduced into unactivated cells and then stimulated to expand in an antigen-specific manner (FIG. 1).

[0610] Patient apheresis derived T cells are enriched via CD4/CD8 microbeads. Following enrichment, the cells are electroporated with a TCR nanoplasmid and separate mRNAs coding for Sleeping Beauty 100X (SB100X) and costimulatory molecules CD70 and CD80. The nonviral transposase-based engineering protocol mediates antigen specific activation to enrich transferase TCR expressing cells. Cells then that express CAR immediately bind to the antigen and are stimulated to survive and expand, enriching engineered cells. The transposon encoding the TCR and molecular enhancers is introduced into the cells prior to activation, allowing for stimulation to occur through the engineered TCR rather than broad T-cell activation. The cells are kept in culture for 2 days in vitro to provide effective antigen specific T cell activation. The in vitro stimulation mediates expansion of infused cells. Example 2. Expression of PRAME InstaTCR Constructs

[0611] FIG. 2A shows experimental data of PRAME TCR expression measured by flow cytometry. Conditions with TCR, SB 100X, peptide, and co- stimulatory molecules generally resulted in both a higher count of viable cells and increased expression of the constructs even 10 days after electroporation (FIG. 2B). Insta TCR cells were kept in extended culture beyond Day 2 with supportive cytokines IL-7 and IL- 15.

[0612] Insta TCR T cells retained TCR expression and expansion potential following cryopreservation. PRAME TCR expression was comparable on Day 6, 10, and 13 post electroporation for no freeze thaw condition and freeze thaw condition as shown in in the flow cytometry plots (FIG. 3A). T cell growth increased comparably in both freeze thaw conditions and no freeze thaw conditions from day of electroporation, through day of freeze, day of thaw, and even 10 days post-thaw (FIG. 3B).

[0613] Dilution of Insta TCR T cells showed continued increase of T cell activation (FIG. 4). A comparison of undiluted and diluted conditions (1: 10 and 1:20) resulted in comparable TCR expression at Days 2, 7, and 14 post-electroporation, indicating that 2-day stimulation is sufficient to drive antigen specific T cell activation.

Example 3. PRAME InstaTCR Constructs Elicit T cell Responses

[0614] In order to determine the effectiveness of Insta TCR, cytotoxicity of Insta TCR T cells and retrovirally engineered TCR T cells was monitored using 3D tumor cell spheroids from SKMEL5 (melanoma) and U2OS (osteosarcoma) cell lines. The cells were monitored for 18 days using Inucyte device. Fresh spheroids were added every 5 days. A control of untransduced cells was also maintained. Mass spectrometry plots showed that Insta T cells expanded significantly better than the RV engineered TCR T cells post tumor cell encounter (FIGs. 5A-B). On day 5, the cells expressing PRAME TCR were enumerated using flow cytometry. The cell counts of Vbl+ cells was much higher in the Insta TCR T cells in comparison to the RV engineered TCR T cells in both the SKMEL5 and U2OS cell lines (FIG. 5C). Insta TCR T cells expand better than he RV engineered TCR T cells post tumor cell encounter.

[0615] A mouse in vitro validation study was developed to determine the effectiveness of TCR T-cells generated through traditional RV and Insta Process, along with a negative control of untransduced cells (FIGs. 6A-D). 624Mel tumor cells were subcutaneously injected into NCG mice after the respective tumor grew to approximately 100mm³ in size. T cells were subsequently injected via i.v. into mice and tumor growth was monitored for 80 days. Insta TCR 10 day-EFl [3e6], which was kept in prolonged culture past day 2, showed decreased tumor volume comparable to RV TCR-MP71 [3e6] cells. Insta TCR 2 day-EFl [3e6] alone did not achieve the decreased tumor volume similar to RV TCR-MP71 [3e6]. Delayed tumor control kinetics of Insta TCR T cells is likely due to time needed for expansion and infusion cells to reach the therapeutic window.

[0616] Flow cytometry plots showing PRAME TCR expression on Day 2, 8, 10, and 14 post electroporation show that the promoter drives efficient TCR expression and T cell expansion. MP71-TCR promoter drives stronger TCR expression on per cell basis in activated T cells while EFla-HTLV-TCR promotes better T cell expansion by quick and early expression of TCR in unactivated T cells (FIGs.7A-B). A study was performed comparing TCR expression and T cell expression using a EFla-HTLV-TCR, a MP71-TCR, and a hybrid promoter MP71-HTLV-TCR. T cell expansion and PRAME TCR expression was monitored on Day 2,5, 8, and 14 using a three-donor study, which found a hybrid promoter, MP71-HTLV-TCR, was found to drive good TCR expression and T cell expansion in the Insta TCR process (FIGs. 8A-B). T cell expansion was monitored on Day 2, 5, 8, and 14 for MP71-TCR and EFla-HTLV conditions. MP71 promoter alone shows decreased cell population compared to EFla-HTLV. When PRAME A02 TCR mRNA was coelectroporated in MP71 promoter Insta TCR, it boosted early expression of TCR on unactivated T cells (FIG. 9). The transient expression of TCR did not compromise specific expansion of TCR T cells.

[0617] Costimulatory molecules mediate effective antigen specific T cell activation in Insta TCR process. CD80 + CD70 together demonstrated the most effectiveness in T cell expansion measured by fold expansion (FIG. 10A). Total TCR expressing T cells were also monitored post electroporation on day 2, 5, 9, and 13. The combination of CD70 + CD80 were found to be the most effective (FIG. 10B). Other costimulatory molecules considered were CD80 and CD70 alone, LIGHT, and D86.

Example 4. Insta TCR process resulted in specific enrichment of HLA A*ll:01/G12V KRAS TCR

[0618] The Insta TCR process also resulted in the most efficient enrichment of HLA A*ll:01/G12V KRAS TCR (FIG. 11). T cells prepared by three different mechanisms were compared. For TCRnp+SB lOOx, T cells were electroporated with HLA A*l l:01/G12V KRAS TCR nanoplasmid and transposase Sleeping Beauty lOOx (SBIOOx). For Pan T cell activation, cells were electroporated with the KRAS TCR and were activated with TransACT post electroporation. The TransACT™ (Miltenyi™ Catalog No. 130-111-160) method was performed according to the manufacture’s protocol. For example, the cells were activated by contacting with the colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists which provides primary and co- stimulatory signals for an optimized and efficient T cell activation and expansion. Lastly, Insta TCR process cells were prepared according to the Insta TCR process described in the present disclosure. For example, the cells were electroporated with HLA A*l l:01/G12V KRAS TCR nanoplasmid, transposase Sleeping Beauty lOOx, costimulatory molecules CD70 and CD80 RNA and pulsed with KRAS minimal epitope. TCR expression of all three cell types was assessed on day 13 for marker CD3.

Table 1 - KRAS TCR Sequences

Table 2 - PRAME-TCR Sequences

Table 3 - Interleukin 2 Receptor Sequences

[0619] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT 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 proteins selected 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 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;

B comprises 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.

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 II wherein 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, or

HLA 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 of an 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 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.

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 10⁴ T 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 10⁸ T 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 IO¹⁰ cells 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 IxlO⁹ immune 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 2xl0⁹ immune 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 10⁶ T 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 10¹⁰ cells.

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, 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.

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