Compositions and methods for treating neoplasia

Abstract

The present disclosure provides engineered T cells comprising TCRs that specifically recognize and bind KRAS G12V, and methods of using such cells for the treatment of neoplasias.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING NEOPLASIA

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority- to and the benefit of U.S. Provisional Application Nos. 63 / 563,841, filed March 11, 2024 and 63 / 692,987, filed September 10, 2024, the entire contents of each of which are incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Kirsten rat sarcoma viral oncogene homologue (KRAS) is commonly mutated in a variety of cancers including colorectal cancer, esophageal cancer, head and neck cancer, lary nx cancer, lung cancer (e.g., non-small-cell lung cancer (NSCLC), small cell lung cancer, squamous cell lung cancer), pancreatic cancers, breast cancer (e.g., triple-negative breast cancer), endometrial cancer, and ovarian cancer (e.g.. high-grade serous ovarian cancer). The most common KRAS mutations observed in cancer are at codons 12 and 13, which promote cellular proliferation and cell survival. Mutations in KRAS are commonly associated with poor outcomes. Accordingly, improved methods for treating tumors having KRAS mutations are urgently required.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides engineered T cells comprising T cell receptors (TCRs) that specifically recognize and bind a Kirsten rat sarcoma viral oncogene homologue (KRAS) G12V mutation in a human leukocyte antigen (HLA) serotype HLA-A*02 context, and methods of using such cells for the treatment of neoplasias.

[0008] In an aspect, the present disclosure provides a binding polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex. The binding polypeptide or an antigen binding portion thereof includes one or more complementarity- determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 and a heterologous constant region.

[0009] In another aspect, the present disclosure provides a T cell receptor polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wildtype KRAS polypeptide: HLA complex. The binding polypeptide or an antigen binding portion thereof includes one or more complementarity determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 and a heterologous constant region.

[0010] In another aspect, the present disclosure provides a polynucleotide encoding the binding polypeptides or the T cell receptors of any of the above aspects, or embodiments thereof.

[0011] In another aspect, the present disclosure provides a vector including the polynucleotide of any of the above aspects, or embodiments thereof.

[0012] In another aspect, the present disclosure provides a cell including the polynucleotide of any of the above aspects, or embodiments thereof, or the vector of any of the above aspects, or embodiments thereof.

[0013] In another aspect, the present disclosure provides a composition including the cell of any of the above aspects, or embodiments thereof.

[0014] In another aspect, the present disclosure provides a kit including the binding polypeptide, the T cell receptor, or an antigen binding portion thereof, of any of the above aspects, or embodiments thereof.

[0015] In another aspect, the present disclosure provides a kit including an isolated polynucleotide of any of the above aspects, or embodiments thereof.

[0016] In another aspect, the present disclosure provides a kit including the composition of any of the above aspects, or embodiments thereof.

[0017] In another aspect, the present disclosure provides a kit including the vector of any of the above aspects, or embodiments thereof, or the cell of any of the above aspects, or embodiments thereof.

[0018] In another aspect, the present disclosure provides an engineered host cell including a heterologous T cell receptor that specifically binds a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex. The heterologous T cell receptor includes: a T cell receptor (TCR) a chain including an amino acid sequence with at least about 85% sequence identity to a TCR a chain amino acid sequence of Table 1; or a TCR P chain including an amino acid sequence with at least about 85% sequence identity to a TCR P chain amino acid sequence of Table 1.

[0019] In another aspect, the present disclosure provides an engineered host cell including a heterologous T cell receptor that specifically binds a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex. The heterologous T cell receptor includes: a TCR a chain comprising complementarity determining region (CDR) 1, CDR2, and CDR3; and a TCR P chain comprising CDR1. CDR2. and CDR3. wherein each CDR comprises an amino acid sequence having at least 85% identity’ to an amino acid sequence listed in Table 2.

[0020] In another aspect, the present disclosure provides an engineered host cell including a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex. The heterologous T cell receptor includes: a T cell receptor (TCR) a chain variable (Va) domain comprising an amino acid sequence with at least about 85% sequence identity' to

[0021] In another aspect, the present disclosure provides an engineered host cell including a heterologous T cell receptor that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, a CD8 co-receptor, and a FAS extracellular domain fused to 41BB intracellular signaling domain and / or an IL7 receptor alpha polypeptide.

[0022] In another aspect, the present disclosure provides an engineered host cell including: a polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex; a CD8 co-receptor; and a FAS extracellular domain fused to 4 IBB intracellular signaling domain and / or an IL7 receptor alpha polypeptide. The polynucleotide encoding the heterologous T cell receptor is inserted at a TRAC, TRBC 1 , or TRBC2 locus of the host cell, the mutant form of KRAS peptide includes a G12V mutation, and the peptide:HLA complex includes an HLA protein encoded by an HLA-A*02 allele.

[0023] In another aspect, the present disclosure provides an engineered host cell including: a heterologous TCR that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex; and a CD8 co-receptor. The heterologous T cell receptor includes a TCR a chain and a TCR P chain of Table 1.

[0024] In another aspect, the present disclosure provides a polynucleotide encoding a heterologous T cell receptor that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex. The heterologous T cell receptor includes: a T cell receptor (TCR) a chain comprising an amino acid sequence with at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to a TCR a chain amino acid sequence of Table 1; or a TCR P chain comprising an amino acid sequence with at least about 85%. at least about 90%. at least about 95%, or about 100% sequence identity to a TCR p chain amino acid sequence of Table 1.

[0025] In another aspect, the present disclosure provides a polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex. The heterologous T cell receptor includes: a TCR a chain framework (FR) 1. complementarity determining region (CDR) 1, FR2, CDR2. FR3. CDR3. or FR4 region or a TCR P chain FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprising an amino acid sequence with at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to a corresponding TCR a chain FR1, CDR1, FR2, CDR2. FR3, CDR3, or FR4 region or a TCR P chain FR1, CDR1, FR2, CDR2. FR3. CDR3. or FR4 region of Table 2.

[0026] In another aspect, the present disclosure provides a polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex. The heterologous T cell receptor includes: a T cell receptor (TCR) a chain variable (Va) domain comprising an amino acid sequence with at least about 85%, at least about 90%, at least about 95, or about 100% sequence identity' to GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQ DQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAETLRDTDKLIFGTGTRLQVFP; or a TCR P chain variable (VP) domain comprising an amino acid sequence with at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLD KSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLVALAIDGELFFGEGSRLTV L, GAVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKAT YEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSADVLAGGTDTQYFGPGTRLT VL, or GAVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKAT YEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAPRWGEQFFGPGTRLTV.

[0027] In another aspect, the present disclosure provides a vector including the polynucleotide of any of the above aspects, or embodiments thereof.

[0028] In another aspect, the present disclosure provides a cell including the polynucleotide of any of the above aspects, or embodiments thereof, or the vector of any of the above aspects, or embodiments thereof.

[0029] In another aspect, the present disclosure provides a pharmaceutical composition including the engineered host cell of any of the above aspects, or embodiments thereof, and a pharmaceutically acceptable excipient.

[0030] In another aspect, the present disclosure provides a method of treating a disease or disorder associated with a KRAS G12V mutation in a subject. The method involves administering to the subject an effective amount of the host cell of any of the above aspects, or embodiments thereof, or the pharmaceutical composition of any of the above aspects, or embodiments thereof.

[0031] In another aspect, the present disclosure provides a method of treating a neoplasia. The method involves administering the engineered host cell of any of the above aspects, or embodiments thereof, to a subject in need thereof.

[0032] In another aspect, the present disclosure provides a kit comprising the host cell of any of the above aspects, or embodiments thereof, or the pharmaceutical composition of any of the above aspects, or embodiments thereof, and instructions for use in a method of treating a neoplasia.

[0033] In another aspect, the present disclosure provides a gene cassette including the polynucleotide sequence of any one of the above aspects, or embodiments thereof, and a polynucleotide sequence encoding a CD8 coreceptor polypeptide and / or a polynucleotide sequence encoding a fusion protein including an intracellular portion of an Interleukin 7 Receptor A (IL7RA) polypeptide.

[0034] In another aspect, the present disclosure provides a cell including the gene cassette of any of the above aspects, or embodiments thereof.

[0035] In another aspect, the present disclosure provides a pharmaceutical composition including the gene cassette of any of the above aspects or embodiments thereof, or the cell of any of the above aspects, or embodiments thereof, and a pharmaceutically acceptable excipient.

[0036] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes TCR 130 a and / or p chains, where the alpha chain includes a CDR1 including TSESDYY; a CDR2 including QEAYKQQN, and a CDR3 including AYNDYKLS; and the beta chain includes a CDR1 including SGHVS, a CDR2 including FQNEAQ, and a CDR3 including ASSLVALAIDGELF.

[0037] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes TCR 240 a and / or P chains, where the alpha chain includes a CDR1 including TRDTTYY; a CDR2 including RNSFDEQN, and a CDR3 including ALSNDYKLS; and the beta chain includes a CDR1 including DFQATT, a CDR2 including SNEGSKA, and a CDR3 including SADVLAGGTDTQY.

[0038] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes TCR 243 a and / or p chains, where the alpha chain includes a CDR1 including DSSSTY; a CDR2 including IFSNMDM, and a CDR3 including AETLRDTDKLI; and the beta chain includes a CDR1 including DFQATT, a CDR2 including SNEGSKA, and a CDR3 including SAPRWGEQF.

[0039] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes a V alpha region including a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity' to the following: AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQ NATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYNDYKLSFGAGTTVTVRA and a V beta region including a sequence having at least about 85%, at least about 90%, at least about 95%. or about 100% amino acid sequence identity to the following: GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLD L.

[0040] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity’ to the following:

[0041] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes a beta chain comprising or consisting of a sequence having at least about 85%. at least about 90%, at least about 95%, or about 100% amino acid sequence identity’ to the following:

[0042] In any of the above aspects, or embodiments thereof, the binding poly peptide or the T cell receptor polypeptide includes a V alpha region including a sequence having at least about 85%. at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:

[0043] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:

[0044] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes a beta chain comprising or consisting of a sequence having at least about 85%. at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

[0045] In any of the above aspects, or embodiments thereof, the binding poly peptide or the T cell receptor polypeptide includes a V alpha region including a sequence having at least about 85%. at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following: V beta region including a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

[0046] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

[0047] In any of the above aspects, or embodiments thereof, the binding polypeptide or the T cell receptor polypeptide includes a beta chain comprising or consisting of a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity’ to the following:

[0048] In any of the above aspects, or embodiments thereof, the binding polypeptide or the antibody, or a binding portion thereof, includes an affinity' tag. In any of the above aspects, or embodiments thereof, the binding polypeptide or the antibody, or a binding portion thereof, includes a detectable amino acid sequence.

[0049] In any of the above aspects, or embodiments thereof, the polynucleotide includes a nucleic acid sequence listed in Table 1 or Table 2.

[0050] In any of the above aspects, or embodiments thereof, the vector is a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector.

[0051] In any of the above aspects, or embodiments thereof, the cell is a mammalian cell. In any of the above aspects, or embodiments thereof, the cell is an immune cell. In any of the above aspects, or embodiments thereof, the cell is a T cell.

[0052] In any of the above aspects, or embodiments thereof, the TCR a chain includes an amino acid sequence with at least about 90% sequence identity to a TCR a chain amino acid sequence of Table 1, or the TCR P chain includes an amino acid sequence with at least about 90% sequence identity to a TCR P chain amino acid sequence of Table 1. In any of the above aspects, or embodiments thereof, the TCR a chain includes an amino acid sequence with at least about 95% sequence identity to a TCR a chain amino acid sequence of Table 1, or the TCR P chain includes an amino acid sequence with at least about 95% sequence identity to a TCR P chain amino acid sequence of Table 1. In any of the above aspects, or embodiments thereof, the TCR a chain amino acid sequence is a TCR a chain amino acid sequence of Table 1, or the TCR P chain amino acid sequence is a TCR P chain amino acid sequence of Table 1. In any of the above aspects, or embodiments thereof, each CDR includes an amino acid sequence having at least about 90% sequence identity to an amino acid sequence listed in Table 2. In any of the above aspects, or embodiments thereof, each CDR includes an amino acid sequence having at least about 95% sequence identity to an amino acid sequence listed in Table 2. In any of the above aspects, or embodiments thereof, each CDR comprises or consists of an amino acid sequence listed in Table 2.

[0053] In any of the above aspects, or embodiments thereof, the T cell receptor (TCR) a chain variable (Va) domain icnludes an amino acid sequence with at least about 90% sequence identity to the TCR P chain variable (VP) domain includes an amino acid sequence with at least about 95% sequence identity to

[0054] In any of the above aspects, or embodiments thereof, the engineered host cell further includes a CD8 co-receptor. In any of the above aspects, or embodiments thereof, the engineered host cell further includes a FAS extracellular domain fused to 41BB intracellular signaling domain and / or an IL7 receptor alpha polypeptide.

[0055] In any of the above aspects, or embodiments thereof, the mutant form of KRAS peptide includes an amino acid sequence of KLVVVGAVGV. In any of the above aspects, or embodiments thereof, the cell is an immune cell. In any of the above aspects, or embodiments thereof, the immune cell is a T cell. In any of the above aspects, or embodiments thereof, the T cell includes a CD4+T cell, a C'D8 T cell, a CD4" CD8" double negative T cell, a yδ T cell, or any combination thereof.

[0056] In any of the above aspects, or embodiments thereof, the engineered host cell further includes a genomic mutation which causes or contributes to decreased expression of endogenous T cell receptor a constant (TRAC), T cell receptor P constant 1 (TRBC1), or a T cell receptor β constant 2 (TRBC2) of the host cell.

[0057] In any of the above aspects, or embodiments thereof, the polynucleotide encoding a heterologous T cell receptor is flanked by a homology arm sequence complementary to a polynucleotide sequence encoding TRAC. TRBC1. or TRBC2. or a homology arm sequence complementary to a polynucleotide sequence having at least 85% polynucleotide sequence identity to a genomic sequence flanking or proximal to a genomic sequence encoding TRAC, TRBC1, or TRBC2.

[0058] In any of the above aspects, or embodiments thereof, the polynucleotide further includes a promoter sequence operably linked to the polynucleotide sequence encoding the heterologous T cell receptor. In any of the above aspects, or embodiments thereof, the promoter sequence is an elongation factor- 1 alpha (EF-la) promoter sequence.

[0059] In any of the above aspects, or embodiments thereof, the vector is a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector.

[0060] In any of the above aspects, or embodiments thereof, the subject is positive for an HLA-A*02 allele. In any of the above aspects, or embodiments thereof, the subject is positive for an HLA-A*02:01 allele.

[0061] In any of the above aspects, or embodiments thereof, the KRAS G12 mutation is a KRAS G12V mutation.

[0062] In any of the above aspects, or embodiments thereof, the disease or disorder is a neoplasia. In any of the above aspects, or embodiments thereof, the neoplasia is a solid cancer. In any of the above aspects, or embodiments thereof, the neoplasia is a hematological malignancy. In any of the above aspects, or embodiments thereof, the neoplasia is selected from the group consisting of: a colorectal cancer, esophageal cancer, head and neck cancer, lary nx cancer, lung cancer, pancreatic cancer, breast cancer, endometrial cancer, and ovarian cancer. In any of the above aspects, or embodiments thereof, the lung cancer is non-smallcell lung cancer, small cell lung cancer, or squamous cell lung cancer. In any of the above aspects, or embodiments thereof, the pancreatic cancer is pancreatic ductal adenocarcinoma. In any of the above aspects, or embodiments thereof, the breast cancer is triple-negative breast cancer. In any of the above aspects, or embodiments thereof, the ovarian cancer is high-grade serous ovarian cancer.

[0063] In any of the above aspects, or embodiments thereof, the cancer is characterized as comprising a KRAS G12V mutation prior to treatment of the subject.

[0064] In any of the above aspects, or embodiments thereof, the subject is selected for treatment by characterizing a biological sample of the subject.

[0065] In any of the above aspects, or embodiments thereof, the subject has been gen o typed for an HLA-A allele prior to the administering.

[0066] In any of the above aspects, or embodiments thereof, the gene cassette includes one or more polynucleotides encoding a self-cleaving peptide flanking one or more of the polynucleotides encoding the TCR a chain, the TCR p> chain, the CD8 coreceptor polypeptide, and the fusion protein including an intracellular portion of the IL7RA polypeptide.

[0067] Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims.

[0068] Definitions

[0069] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill \\ i th a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology' and Molecular Biology' (2nd ed. 1994); The Cambridge Dictionary' of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed.. R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary / of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0070] By “agent’" is meant a polypeptide, polynucleotide, a cell engineered to express a heterologous polypeptide or polynucleotide, or a small compound. In some embodiments, the cell is an immune cell (e.g., T cell) that is autologous or heterologous to a subject. In one embodiment, the agent is a T cell expressing a T cell receptor (TCR) that binds a Kirsten rat sarcoma viral oncogene homologue (KRAS) polypeptide comprising a mutation (e.g., a G12V mutation). By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In one embodiment, the disease is cancer (e.g.. a cancer featuring a KRAS G12V mutation).

[0071] By “alteration” is meant a change (increase or decrease) in the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. In one embodiment, the alteration is a G12V mutation in a KRAS polypeptide. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels. “

[0072] By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include any unnatural amino acid.

[0073] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0074] As used herein, the term “CD8 co-receptor polypeptide" or "CD8 polypeptide " is meant cell surface glycoprotein CD8 or a fragment thereof. CD8 is present at the cell surface as either as an CD8alpha subunit-CD8alpha subunit homodimer or a CD8alpha subunit- CD8beta subunit heterodimer. The CD8 co-receptor assists in the function of cytotoxic T cells (CD8+) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen. Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1 :81-88, 2004). There are five (5) documented human CD8 beta chain isoforms (see UniProtKB identifier Pl 0966) and a single documented human CD8 alpha chain isoform (see UniProtKB identifier P01732). Methods for characterizing the presence of CD8 on the surface of a cell are known in the art and described in Gao supra and Cole supra.

[0075] By “CD8alpha polypeptide” or “CD8a polypeptide” is meant a polypeptide having at least about 85% amino acid sequence identity to NCBI Accession Nos. NP_001759.3 or NP_741969.1, or a fragment thereof having co-receptor activity for ligand recognition by T cell receptors when dimerized with another CD8a or a CD8p. Exemplary CD8a amino acid sequences are provided below: >NP_001759.3 T-cell surface glycoprotein CD8 alpha chain isoform 1 precursor [Homo sapiens]

[0076] >NP_741969.1 T-cell surface glycoprotein CD8 alpha chain isoform 2

[0077] By “CD8alpha polynucleotide'’ or “CD8a polynucleotide'’ is meant a nucleic acid molecule encoding a CD8a polypeptide. Exemplary CD8alpha polynucleotide sequence are provided below:

[0078] >NM_001768.7 Homo sapiens CD8 subunit alpha (CD8A) , transcript variant 1, mRNA ,

[0079] By “CD8beta polypeptide” or “CD8[3 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession Nos. XP_054200532. 1, NP_757362. 1 , NP_742099. 1 , NP_742100. 1 , NP_004922.1 , or NP_001171571 . 1 , or a fragment thereof having co-receptor activity for ligand recognition by T cell receptors when dimerized with a CD8a. Exemplary CD8P amino acid sequences are provided below: >XP_054200532 . 1 T- cell surface glycoprotein CD8 beta chain isoform XI [Homo sapiens]

[0080] >NP_757362 . 1 T- cel l surface glycoprotein CD8 beta chain isoform 2 precursor [Homo sapiens] >NP_742099.1 T-cell surface glycoprotein CD8 beta chain isoform 3 precursor [Homo sapiens]

[0081] >NP_742100.1 T-cell surface glycoprotein CD8 beta chain isoform 4 precursor

[0082] >NP_004922.1 T-cell surface glycoprotein CD8 beta chain isoform 5 precursor [Homo sapiens]

[0083] >NP_001171571.1 T-cell surface glycoprotein CD8 beta chain isoform 6

[0084] By “CD8beta polynucleotide” or “CD8P polynucleotide” is meant a nucleic acid molecule encoding a CD8P polypeptide.

[0085] >NM_004931.5 Homo sapiens CD8 subunit beta (CD8B) , transcript variant 5, mRNA

[0086] >NM_172213.5 Homo sapiens CD8 subunit beta (CD8B) , transcript variant 2,

[0087] >NM_001178100.2 Homo sapiens CD8 subunit beta (CD8B) , transcript variant 6, G A v

[0088] A

[0089] A

[0090] G

[0091] A

[0092] G

[0093] A

[0094] A

[0095] G

[0096] A

[0097] A

[0098] A

[0099] A

[0100] A

[0101] G

[0102] G

[0103] A

[0104] The terms "complementarity determining region," and "CDR." generally refer to sequences of amino acids within immunoglobulin superfamily member (e g., TCR) variable regions, which confer antigen specificity or binding affinity' and are separated from one another in primary amino acid sequence by framework regions (FRs). In general, there are three CDRs in each TCR a-chain variable region (aCDRl, aCDR2. aCDR3) and three CDRs in each TCR P-chain variable region (PCDR1, PCDR2, PCDR3). In TCRs, CDR3 is thought to be the main CDR responsible for recognizing processed antigen. In general, CDR1 and CDR2 interact mainly or exclusively with the MHC.

[0105] CDR1 and CDR2 are encoded within the variable gene segment of a TCR variable region-coding sequence, whereas CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for Vp. Thus, if the identity of the variable gene segment of a Va or VP is described, the sequences of their corresponding CDR1 and CDR2 can be deduced; e.g., according to a numbering scheme as described herein. Compared with CDR1 and CDR2. CDR3 can be significantly more diverse due to the addition and loss of nucleotides during the recombination process.

[0106] TCR variable domain sequences can be aligned to a numbering scheme (e g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using, for example, ANARCI software tool (2016, Bioinformatics 15:298-300). A numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains. In certain embodiments, a CDR of the present disclosure is identified according to the IMGT numbering scheme or method (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, a CDR of the present disclosure is identified according to the Kabat numbering scheme or method. In some embodiments, a CDR of the present disclosure is identified according to the Chothia numbering scheme or method. In some embodiments, a CDR of the present disclosure is identified according to the EU numbering scheme or method. In some embodiments, a CDR of the present disclosure is identified according to the enhanced Chothia numbering scheme or method. In some embodiments, a CDR of the present disclosure is identified according to the Aho numbering scheme or method.

[0107] By “decreases’" is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%. 10%. 15%. 20%. 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages.

[0108] “Detect” refers to identifying the presence, absence, or amount of the analyte to be detected.

[0109] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is a cancer associated with an alteration in a Kirsten rat sarcoma viral oncogene homologue (KRAS) polypeptide (e.g., KRAS G12V) or in a polynucleotide encoding said polypeptide. In embodiments, a cancer associated with a KRAS mutation (e.g., KRAS G12V) is a pancreatic cancer (e.g., pancreatic ductal adenocarcinoma) colorectal cancer, esophageal cancer, head and neck cancer, larynx cancer, lung cancer (e.g., small cell lung cancer, squamous cell lung cancer), breast cancer (e.g., triple-negative breast cancer), endometrial cancer, and ovarian cancer (e.g., high-grade serous ovarian cancer). In an embodiment, the disease is in a subject expressing human leukocyte antigen (HLA) serotype HL A-A*02.

[0110] The term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0111] By "effective amount" is meant the amount of required to ameliorate a symptom of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0112] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0113] As used herein, a(n) "heterologous" or "exogenous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule, or portion of a or nucleic acid molecule that is not native to a host cell but can be homologous to a nucleic acid molecule or portion thereof from the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule (i.e., not endogenous or native) is added to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector) and can be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein, polypeptide, or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity. Moreover, a cell comprising a "modification” or a "heterologous" polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were themselves transduced, transfected, or otherwise manipulated or changed.

[0114] A “host cell” or “cell” is any prokaryotic or eukary otic cell that contains either a cloning vector or an expression vector. This term also includes those prokary otic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. In some embodiments, the host cell is an immune cell (e.g.. T cell, macrophage, natural killer cell). In some embodiments, the host cell is an induced pluripotent stem (iPS) cell, or a derivative thereof. iPS host cells of the present disclosure may be modified before, during, and / or after differentiation. In some embodiments, the host cell comprises a vector comprising a polynucleotide encoding a heterologous TCR of the present disclosure. In some embodiments, the host cell expresses a heterologous TCR of the present disclosure.

[0115] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0116] As used herein, an "immune cell" generally refers to any cell of the immune system. In embodiments, an immune cell originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y5 T cell, a regulatory T cell, a natural killer cell, a natural killer T cell, and a dendritic cell. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs," which are specialized immune cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[0117] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is. a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0118] By "isolated polynucleotide" is meant a nucleic acid molecule (e.g., a DNA, RNA) that is free of the genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0119] By an "isolated polypeptide" is meant a polypeptide that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0120] By “marker” is meant any protein, polynucleotide, clinical indicator, or other analyte having an alteration that is associated with a disease or disorder.

[0121] By “operably^ linked” refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. The described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means positioning the coding sequence such that the expression of the coding sequence is controlled by the promoter.

[0122] By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19. 20, or 21 nucleotides.

[0123] By "‘positioned for expression’’ is meant that the polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a TCR described herein).

[0124] The term '‘promoter” as used herein refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be “inducible”, initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does not regulate the rate of transcription. A promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types. In some embodiments, the promoter is an endogenous promoter (e.g., TRAC or TRBC promoter or the promoter that drives the expression of a gene located in a genomic safe harbor). In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is an elongation factor- 1 alpha (EF-la) promoter. In some embodiments, the promoter is a Meiotic Nuclear Divisions 1 (MND1) promoter.

[0125] By “RNA-seq” is meant RNA sequencing for detecting and quantifying messenger RNA molecules (mRNA) in a biological sample, which, for example, may be used to study cellular responses. A related term, “scRNA-seq” is single-cell RNA sequencing, which may be, for example, a droplet-based single-cell RNA-seq or “Drop-seq,” that is a sequencing technology for analyzing RNA expression in at least hundreds of thousands of individual cells in embodiments of the disclosure, but may alternatively use any other high-throughput sequencing platform.

[0126] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0127] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0128] By “reference” is meant a standard or control condition. In some embodiments, the reference is a healthy subject or cell. In some embodiments, the reference is an untreated subject or cell having a disease (e.g., a subject having aneoplasia or a neoplastic cell). In some embodiments, the reference is a KRAS wildtype peptide, or KRAS peptide not comprising a G12V mutation. A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides or at least about 300 nucleotides or any integer thereabout or therebetween.

[0129] By "specifically binds" is meant a polynucleotide or polypeptide that recognizes and binds a target polypeptide or polynucleotide, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptides or polynucleotides described herein. In some embodiments, T cell receptors (TCRs) of the present disclosure specifically bind a KRAS polypeptide comprising a G12V mutation, or a fragment thereof comprising the G12V mutation, and fail to detectably bind or bind at significantly lower levels to KRAS polypeptides not including said mutation.

[0130] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity" to an endogenous sequence are Wpically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0131] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, and less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, and of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In an embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In an embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate. 1% SDS. 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In an embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0132] For most applications, washing steps that follow' hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, and less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the w'ash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, and of at least about 68° C. In an embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In an embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1 .5 mM trisodium citrate, and 0.1 % SDS. In an embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In an embodiment, such a sequence is at least 60%, more preferably 80% or 85%, and 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0133] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BUAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions ty pically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary’ approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0134] By "subject" is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline. In some embodiments, the subject is a human subject.

[0135] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29. 30. 31. 32. 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0136] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0137] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0138] As used herein "T cell" or "T lymphocyte" generally refers to an immune cell that matures in the thymus and produces a T cell receptor (TCR). T cells can be naive ("TN"; not exposed to antigen; increased expression of CD62L, CCR7. CD28, CD3, CD127, and CD45RA, and decreased or no expression of CD45RO as compared to TCM (described herein)), memory T cells (TM) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM, express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to TCM, and are positive for granzyme and perforin. Helper T cells (TH) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals. T cells can be collected using suitable techniques, and the various subpopulations or combinations thereof can be enriched or depleted by suitable techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28-, and Qa-1 restricted T cells.

[0139] As used herein, a "T cell receptor" (TCR) generally refers to an immunoglobulin superfamily member comprising a variable binding domain, a constant domain, and optionally a transmembrane region, and a short cytoplasmic tail that is capable of specifically binding to an antigen peptide bound to an MHC receptor. See, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997. A TCR can be found on the surface of a cell or in soluble form and generally comprises heterodimer having a and P chains (also known as TCR a and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively). In some embodiments, the TCR specifically binds a KRAS peptide having a mutation (e.g., a G12V mutation). In some embodiments, the TCR specifically binds a peptide bound to an HLA-A*02 MHC molecule (e.g., an HLA-A*02:01 MHC molecule).

[0140] The term "variable region" or "variable domain" generally refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR a-chain or P-chain (or y chain and 5 chain for y5 TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g.. TCR) to antigen. The variable domains of the a chain and P chain (Va and VP, respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. The Va domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the VP domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single Va or VP domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind a particular antigen may be isolated using a Va or VP domain from a TCR that binds the antigen to screen a library of complementary Va or VP domains, respectively.

[0141] By ‘‘vector” is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell. In one embodiment, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers. In some embodiments, the vector is a lentiviral vector.

[0142] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0143] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0144] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0145] BRIEF DESCRIPTION OF THE DRAWINGS

[0146] FIG. 1 provides graphs showing that high transduction efficiency was achieved for both Batch 1 and Batch 2 of transduced T cells. Each batch of TCRs was transduced into T cells using a lentiviral vector that included a polynucleotide encoding TCRa / p chains from a TCR of interest, and CD8a / p polypeptides, each separated with 2A self-cleaving peptides. Batch 1 was evaluated with both mouse and human constant regions for the TCR, and Batch 2 was only evaluated with human constant regions for the TCR. A star indicates the TCR from each batch chosen for further analysis (i.e., TCR #s 130 and 240). UTD = untreated. FIG. 2 provides graphs showing that transduced T cells from Batch 1 were able to recognize a peptide-major histocompatibility complex (MHC) tetramer. The tetramer was made from four subunits, each subunit including a human leukocyte antigen (HLA) serotype HLA-A*02 / peptide of interest complex. The peptide of interest was a polypeptide fragment of KRAS containing the G12V mutation. A star indicates the TCR. UTD = untreated; MFI = mean fluorescence intensity.

[0147] FIG. 3 provides graphs showing that the TCR chosen for further analysis from Batch 1 displayed high sensitivity for KRAS G12V peptide. T cells from 2 different donors were transduced with the TCR chosen from Batch 1 (TCR 130). The transduced TCR 130 T cells were co-cultured with T2 cells loaded with KRAS G12V peptide. Interferon (IFN)y response was subsequently measured.

[0148] FIG. 4 provides graphs showing that the TCR chosen for further analysis from Batch

[0149] 1 showed cytotoxicity in multiple tumor models. T cells transduced with TCR 130 were introduced to cultures containing pancreatic adenocarcinoma cell models (CFPAC1, DANG. PATU8902) and colorectal adenocarcinoma cell models (SW480, SW527, SK-CO1) expressing KRAS G12V peptide and endogenous HLA-A*02. Tumor cell confluence was measured over time. TCR130 was the most effective TCR tested in reducing tumor cell confluence.

[0150] FIG. 5 provides graphs showing that TCRs from Batch 1 were specific for cells expressing both HLA-A*02 and a KRAS G12V peptide. In the absence of both HLA-A*02 and a KRAS G12V no cytotoxicity was observed. T cells transduced with TCR 130 were introduced to HuCCTl cells expressing HLA-A*l l:01 and both of TP53 R175H and KRAS G12D peptides, as well as Tyknu cells expressing HLA-A*02:01 / 26:03 and both of TP53 R175H and KRAS wild type peptides. Tumor cell confluence was measured.

[0151] FIG. 6 provides graphs showing that transduced T cells from Batch 2 recognized a peptide-maj or histocompatibility’ complex (MHC) tetramer. The tetramer was made from four subunits, each subunit including a human leukocyte antigen (HLA) serotype HLA- A*02 / peptide of interest complex. The peptide of interest was a polypeptide fragment of KRAS containing the G12V mutation. A star indicates the TCRs chosen for further analysis. UTD = untreated; MFI = mean fluorescence intensity.

[0152] FIG. 7 provides graphs showing that the TCRs chosen for further analysis from Batch

[0153] 2 displayed high sensitivity for KRAS G12V peptide. T cells from 2 different donors were transduced with the TCRs from Batch 2 (TCRs 240 and 243). The transduced TCR 240 / 243 T cells were co-cultured with T2 cells loaded with KRAS G12V peptide. Interferon (IFN)y response was subsequently measured.

[0154] FIG. 8 provides graphs showing that the TCRs chosen for further analysis from Batch 2 displayed cytotoxicity in multiple tumor models. T cells transduced with TCRs 240 and 243 were introduced to pancreatic adenocarcinoma cell models (CFPAC1, DANG) and colorectal adenocarcinoma cell models (SW527, SK-CO1) expressing KRAS G12V peptide and endogenous HLA-A*02. Tumor cell confluence was measured over time in the presence of the TCR.

[0155] FIG. 9 provides a graph showing that high transduction efficiency was achieved for each of the TCRs chosen for further analysis from each of Batch 1 and 2. +ILR indicates that the host cell was further modified to express an interleukin 7 receptor (IL7R) fusion protein (CD34-IL7R)

[0156] FIG. 10 provides graphs showing that the TCR (TCR 130) chosen for further analysis from Batch 1 was able to kill tumor cells even after multiple re-challenges. T cells transduced with TCR 130 and CD8a / 0 co-receptor polypeptide were introduced to SW527 or CFPAC1 cells expressing HLA-A*02 and KRAS G12V polypeptide. Tumor confluence was measured and compared with tumor cells alone (Tumor Only) or untreated T cells (UTD). The black arrows indicate re-challenge. TCR130-ILR indicates that the host cell was further modified to express an IL7R fusion protein (CD34-IL7R).

[0157] FIG. 11 provides graphs showing that the TCRs (TCRs 240 / 243) chosen for further analysis from Batch 2 were able to kill tumor cells even after multiple re-challenges. T cells transduced with TCR 240 / 243 and CD8a / 0 co-receptor polypeptide were introduced to SK- CO-1 or SW480 cells expressing HLA-A*02 and KRAS G12V polypeptide. Tumor confluence was measured and compared with tumor cells alone (Tumor Only) or untreated T cells (UTD). The black arrows indicate re-challenge.

[0158] FIG. 12 provides graphs showing that the TCRs (TCRs 240 / 243) chosen for further analysis from Batch 2 were able to kill tumor cells even after multiple re-challenges. T cells transduced with TCR 240 / 243 and CD8oc / p co-receptor polypeptide were introduced to CFPAC1, DANG, and SW527 cells expressing HLA-A*02 and KRAS G12V polypeptide. Tumor confluence was measured and compared with tumor cells alone (Tumor Only) or untreated T cells (UTD). The transduced host cells in the second row of graphs are further modified to express an IL7R fusion protein (CD34-IL7R). The black arrows indicate rechallenge. FIGs. 13A-13B provide a graph and heatmaps showing that the TCRs chosen for further analysis from Batch 2 were specific for KRAS G12V peptide, with only very limited recognition of wildtype KRAS peptide, or potential off-targets. FTG. 13A, T cells from 2 different donors were transduced with the TCRs from Batch 2 (TCRs 240 and 243). The transduced TCR 240 / 243 T cells w ere co-cultured with T2 cells loaded with KRAS wildtype peptide. Interferon (IFN)y response was subsequently measured. The y-axis is split to emphasize the very limited response shown. FIG. 13B, a KRAS peptide w as individually mutated at each position to each of the 19 different amino acids and each mutated KRAS peptide thus produced was loaded into T2 cells. Each position in the peptide is indicated by the x-axis, and the specific mutation is shown by the y-axis. T cells transduced with TCRs 240 and 243 were introduced to the T2 cells, and IFNy response was subsequently measured.

[0159] FIG. 14 provides a schematic showing a mechanism for non- viral gene editing of T cells w ith TCRs of the present disclosure (KRAS G12V TCRs). The knock-in construct shown is inserted into the T cell receptor a constant (TRAC) locus while the T cell receptor p constant gene is simultaneously knocked out.

[0160] FIG. 15 is a graph showing the percentage of T cells expressing the self-cleaving peptide 2A. This peptide is encoded in a nucleic acid construct between each additional parameter (i.e., an exogenous TCR (i.e., TCR130, TCR240, or TCR243), CDaP co-receptor polypeptide, and an ILR signaling polypeptide encoded on the construct. 2A expression indicates the nucleic acid construct was successfully knocked-in to a target cell genome. “Control” denotes T cells that do not express an exogenous TCR.

[0161] FIG. 16 is a graph showing tetramer binding of the T cells described in FIG. 15. MFI = mean fluorescence intensity.

[0162] FIG. 17 includes graphs showing T cell activation as measured by IFNy expression of T cells derived from three different donors and modified to express a knocked-in exogenous TCR (i.e., TCR130, TCR240, or TCR243), CDaP co-receptor polypeptide, and an ILR signaling polypeptide. “Control” denotes T cells that do not express an exogenous TCR. “dKO” denotes a double knockout of endogenous TRAC and TRBC.

[0163] FIGs. 18A-18C characterize T cells modified via nonviral knock-in to express an exogenous TCR (TCR130, TCR240, and TCR243) and an ILR polypeptide. FIG. 18A is a graph showing IFNy secretion from TCR-T cells or control T cells (i.e., no heterologous TCR) after 7 days of co-culture with HLA- and antigen-positive cell lines. FIG. 18B is a graph quantifying TCR-T proliferation after 7 days of co-culture with HLA- and antigenpositive cell lines. FIG. 18C includes graphs showing cytotoxic activity of T cells nonvirally modified to express a knocked-in exogenous TCR and an ILR polypeptide against HLA- A2+ / KRAS G12V+ CFPAC1. SW527, SKCO1, DAN-G cancer cells and antigen-negative Tyk-nu cancer cells. As used in these figures, “Control’’ denotes T cells that do not express a exogenous TCR. “dKO” denotes a double knockout of endogenous TRAC and TRBC.

[0164] FIG. 19 provides schematics and pictures showing the CRISPR-based insertion of a non-viral knock-in gene cassette into the TRAC locus, and illustrating the production of transgenic T cells designed to target KRAS G12V presented in a HLA-A*02:01 context, which were produced using a non-viral knock-in gene cassette.

[0165] FIG. 20 provides a graph and a chart showing the recognition of exogenous G12V peptide in an HLA-A*02:01 context by engineered TCR-T cells. TCR-T cells engineered to recognize A2-G12V epitope (results for TCR 1 shown) bound HLA-A*02:01 tetramer reagent loaded with G12V peptide (left panel). TCR-T cells showed potent functional response to antigen presenting T2 cells pulsed with G12V peptide, but did not react to T2 cells pulsed with WT KRAS peptide (right panel). The bars in the right panel are provided in two sets of three, and are ordered in each set, from left to right, TCR 1, TCR 2. and TCR 3. TCR 1 is also disclosed herein as TCR 130, TCR 2 is also disclosed herein as TCR 240, and TCR 3 is also disclosed herein as TCR 243.

[0166] FIG. 21 provides charts showing that engineered HLA-A*02:01 cell lines processed and presented G12V. HLA-A*02:01 positive cell lines endogenous for KRAS G12V were transfected with a lentivirus encoding a tandem minigene (TMG) of the G12V peptide (left panel) to overexpress the G12V peptide. HLA-A*02:01 positive cell lines negative for KRAS G12V were also transfected with an mRNA encoding full length KRAS G12V or a TMG of the G12V peptide (right panel). A2-G12V TCR-T cells (or T cells not expressing an A2-G12V TCR -No TCR) were then cocultured with the engineered cell lines. Increased functional response demonstrated the presentation of mutant KRAS G12V peptides by HLA- A*02:01. In the left panel, bars are provided in sets of four and are ordered, from left to right, No TCR, TCR 1, TCR 2, and TCR 3. TCR 1 is also disclosed herein as TCR 130, TCR 2 is also disclosed herein as TCR 240. and TCR 3 is also disclosed herein as TCR 243. In the right panel, the bars are provided in sets of two, and ordered, from left to right. No TCR and TCR 130.

[0167] FIG. 22 provides graphs showing that engineered TCR-T cells were cytotoxic to cell lines with endogenous HLA-A*02:01 and KRAS G12V. TCR-T cells were screened against cell lines endogenously HLA-A*02:01 positive (CFPAC1 cells, top panel; SW527 cells, middle panel) and harboring mutant KRAS G12V mutations. Cytotoxic function was observed against cell lines, indicating processing and presentation of peptide in unmodified cell lines. TCRs did not kill antigen negative Tyk-nu cells (bottom panel). TCR 1 is also disclosed herein as TCR 130, TCR 2 is also disclosed herein as TCR 240, and TCR 3 is also disclosed herein as TCR 243.

[0168] FIG. 23 provides graphs and charts showing that off-target assessment indicated TCR specificity. X-Scan of TCRs resulted in search motif which yielded minimal potential off- target list. Peptide screen identified two potential off-targets with EC50 similar to index peptide. The two potential off-targets (Potential off-target 1 and Potential off-target 2) were tested in a titration for functional response (left panel). Next, TCR 130 was then cocultured with cells overexpressing potential off-targets or a TMG of the G12V peptide and assessed for functional response. Lack of functional response indicated that the potential off-target peptides were not processed and recognized by TCR 130. In the right panel, bars are provided in sets of two, and ordered, from left to right, No TCR and TCR 130.

[0169] DETAILED DESCRIPTION OF THE INVENTION

[0170] The disclosure features compositions comprising engineered T cells and methods of using such cells for treating neoplasias.

[0171] The disclosure is based, at least in part, on the discovery that engineered T cells expressing a TCR that specifically binds a KRAS G12V polypeptide can be used to kill neoplastic cells. Advantageously, these T cells are also engineered to express a CD8 coreceptor (e.g., CD8aP) that acts as co-receptor during T cell antigen engagement enhancing T cell activation signals and a FASBB switch receptor that converts inhibitory7signals to activating or proliferatory signals (e.g., FAS extracellular domain fused to 4-1BB intracellular signalling domain) and / or a cytokine (e.g., IL-7) modulator. In embodiments, the engineered T cell disclosed herein comprises modifications that improve T cell persistence, reduce and / or prevent T cell exhaustion, and / or that reduce the engineered T cells' immunogenicity.

[0172] Adoptive cellular therapies (ACT) have had limited success in the treatment of solid tumors. Recent approvals of both Tumor Infiltrating Lymphocyte (TIL) and T cell receptor- engineered T cell (TCR-T) therapies herald the promise of autologous T cell strategies. Unfortunately, existing therapeutics face significant challenges including an existing T cell response to immunogenic tumors (TILs) or relying on targets lacking tumor dependence (TCR-T). Accordingly, the present disclosure provides T cell therapies utilizing TCRs specific for public oncogenic driver mutations that are designed to overcome these hurdles and drive potent and durable immune responses.

[0173] Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS)

[0174] Kirsten rat sarcoma viral oncogene homologue (KRAS) is the best-known oncogene with the highest mutation rate among all cancers and is associated with a series of highly fatal cancers, including pancreatic ductal adenocarcinoma (PDAC), nonsmall-cell lung cancer (NSCLC), and colorectal cancer (CRC). Mutated forms of the KRAS gene are prevalent in pancreatic carcinomas (>80%), colon carcinomas (40-50%), and lung carcinomas (30-50%), but are also present in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia and breast cancer.

[0175] The KRAS gene encodes the KRAS protein, a GTP / guanosine diphosphate (GDP)- binding protein that belongs to the guanosine triphosphatase (GTPase) RAS family. Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states. Constitutively active KRAS leads to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors.

[0176] Single residue alterations in KRAS are the most frequent oncogenic driver mutations in solid tumors, with KRAS mutations arising in indications with high unmet medical need such as PDAC. CRC, and NSCLC. Accordingly, in some embodiments, the present disclosure provides compositions and methods targeting KRAS mutations (e.g., KRAS G12V mutations).

[0177] Host cells or compositions

[0178] The human leukocyte antigen (HLA) serotype HLA-A*02:01 is the most frequently occurring HLA class T allele in North American and Western European populations. However, therapeutics designed to provoke immunogenic responses to KRAS mutations (e.g., KRAS G12V mutations) presented by HLA-A*02:01 have proven challenging thus far, and physical evidence of the presentation of KRAS mutations (e.g.. the KRAS G12V peptide) by HLA-A*02:01 has been limited. Thus, the present disclosure includes embodiments that provide improved compositions and methods for targeting the mutant KRAS peptides (e.g., KRAS G12V) presented in the context of HLA-A*02:01 that address this urgent need.

[0179] In some aspects, the present disclosure provides for a host cell comprising an extracellular binding protein w herein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises a KRAS G12 mutant peptide. In some embodiments, the peptide:HLA complex comprises an HLA-A*02 allele. In some embodiments, the peptide:HLA complex comprises an HLA-A*02:01 allele. In some embodiments, the peptide:HLA complex comprises an HLA allele that binds or is predicted to bind a KRAS mutant peptide (e.g., the G12 mutant peptide, such as G12V) with a suitable affinity for presentation and TCR activation, for example, a binding affinity or KD of at most 1000 nM, at most 750 nM. at most 500 nM, at most 250 nM, at most 100 nM, at most 50nM, or at most 10 nM.

[0180] In some cases, the host cell comprises an immune cell or a precursor thereof. In some cases, the immune cell comprises a T cell, aNK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In some cases, the immune cell is a T cell, wherein the T cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a CD4+ CD8+ double positive T cell, a y5 T cell, or any combination thereof. In some cases, the host cell further comprises a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a (CD8a) chain or a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor |3 (CD8P) chain. In some cases, when the host cell is in the presence of a tumor cell that expresses a KRAS G12V mutant peptide, CD137 expression of the host cell is elevated as compared to: (i) CD137 expression by a reference human T cell not expressing the binding protein, when the reference human T cell is in the presence of the tumor cell; or (ii) CD137 expression by the human T cell expressing the binding protein when not in the presence of the tumor cell or when not in the presence of an antigen-presenting cell expressing a peptide:HLA complex.

[0181] Host cells

[0182] The disclosure provides host cells (e g., engineered immune cells) and populations thereof that comprise, encode, and / or are capable of expressing an extracellular binding protein disclosed herein.

[0183] A host cell can be a peripheral blood mononuclear cell (PBMC). A host cell can be a lymphoid cell. A host cell can be a lymphocyte. A host cell can be a T cell. A host cell can be a B cell. A host cell can be a natural killer (NK) cell. A host cell can be a Natural Killer T (NKT) cell. A host cell can be a mammalian cell. A host cell can be a human cell.

[0184] A host cell can be a primary cell. A host cell can be an immortalized cell. A host cell can be of a cell line. A host cell can be differentiated from a stem cell, for example, an induced pluripotent stem cell (iPSC), embryonic stem cell, hematopoietic stem cell (HSC). or the like.

[0185] In some cases, the host cells are “off-the-shelf’ cells that are engineered from an immune cell line, for instance, a T cell line or an NK cell line (e.g., NK-92, or e.g., NK-YS, KHYS-1, NKL, NKG, SNK-6, or IMC-1). Such host cells can be readily available and formulated for direct administration to a subject in need thereof.

[0186] A host cell can be allogenic to a subject or patient. In some embodiments, a host cell is autologous to a subject or patient. For example, immune cells (e.g., T cells) can be obtained from a subject or patient and modified ex vivo to generate a host cell comprising a TCR that specifically binds a KRAS G12V peptide.

[0187] A host cell can be an alpha beta T cell (i.e., expressing a TCR comprising alpha and beta chains). A host cell can be a gamma delta T cell (i.e., expressing a TCR comprising gamma and delta chains). In some embodiments, a host cell comprises a disruption or deletion of one or more endogenous TCR-encoding genes, such as TRAC, TRB (e.g., TRBC1 and / or TRBC2), TRG, and / or TRD. In some embodiments, a host cell comprises a disruption or deletion of a variable region of one or more endogenous TCR-encoding genes, such as a disruption or deletion in TRAC, TRB, TRG, and / or TRD. In some embodiments, a host cell comprises a disruption or deletion of a constant region of one or more endogenous TCR- encoding genes.

[0188] Extracellular binding protein

[0189] In some aspects, the present disclosure provides for an extracellular binding protein wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises a KRAS G12 mutant peptide. In some embodiments, the peptide:HLA complex comprises an HLA-A*02 allele. In some embodiments, the peptide:HLA complex comprises an HLA-A*02:01 allele. In some embodiments, the peptide:HLA complex comprises an HLA allele that binds or is predicted to bind a KRAS mutant peptide (e.g., a G12 mutant peptide, such as G12V) with a suitable affinity for presentation and TCR activation, for example, a binding affinity or KD of at most 1000 nM, at most 750 nM, at most 500 nM, at most 250 nM, at most 100 nM, at most 50nM, or at most 10 nM.

[0190] The extracellular binding protein can comprise a TCR or a portion thereof. In some embodiments, the extracellular binding protein comprises a T cell receptor (TCR) a chain variable (Va) region, a TCR P chain variable (VP) region, a T cell receptor (TCR) a chain constant (Ca) region, and / or a T cell receptor (TCR) P chain constant (CP) region. The extracellular binding protein can also comprise a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1. CDR1. FR2. CDR2. FR3, CDR3, or FR4 region; or a TCRP FR1, CDR1 , FR2, CDR2, FR3, CDR3, or FR4 region, or a combination thereof.

[0191] The extracellular binding protein may comprise (i) an extracellular domain of TCR alpha chain constant region, TCR beta chain constant region, TCR gamma chain constant region, or TCR delta chain constant region; (ii) a transmembrane domain of TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain; and / or (hi) a cytoplasmic domain of TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain. The extracellular binding protein can comprise a full length or substantially full length TCR alpha chain, TCR beta chain, TCR gamma chain, and / or TCR delta chain.

[0192] In some cases, the extracellular binding protein comprises a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRP FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprising a sequence having at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%. at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any of the TCR Va, VP, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences described in Table 1.

[0193] An extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRP FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%. at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%. at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%. at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity' or sequence similarity to any one of the TCR Va, VP, FR1, CDR1, FR2, CDR2, FR3. CDR3, or FR4 sequences disclosed in Table 1. An extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1. CDR1. FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRP FR1 , CDR1 , FR2, CDR2, FR3, CDR3, or FR4 region disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at most about 70%, at most about 71%. at most about 72%, at most about 73%, at most about 74%, at most about 75%, at most about 76%. at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about 88%, at most about 89%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 95.5%, at most about 96%, at most about 96.5%. at most about 97%. at most about 97.5%, at most about 98%, at most about 98.5%, at most about 99%, or at most about 99.5% sequence identity or sequence similarity to any one of the TCR Va, VP, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences disclosed in Table 1.

[0194] In some embodiments, an extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3. CDR3, or FR4 region; or a TCRP FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises, consists essentially of, or consists of an amino acid sequence with about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%. about 77%, about 78%, about 79%. about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%. about 87%, about 88%, about 89%. about 90%. about 91%. about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 100% sequence identity or sequence similarity to any one of the TCR Va, VP, FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 sequences disclosed in Table 1.

[0195] The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs designed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe. Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm. A global alignment algorithm, such as a Needleman and Wunsch algorithm, can be used to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Default settings can be used. To generate similarity scores for two amino acid sequences, scoring matrices can be used that assign positive scores for some non-identical amino acids (e.g.. amino acids with similar physio-chemical properties and / or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs). Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0196] In some embodiments, the extracellular binding protein, a TCR a chain variable (V a) domain; a TCR β chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises, consists essentially of, or consists of the amino acid sequence of any one of the TCR Va, VP, FR1. CDR1. FR2. CDR2. FR3, CDR3, or FR4 sequences disclosed in Table 1.

[0197] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence with one or more insertions, deletions, and / or substitutions relative to any one of the sequences disclosed in Table 1.

[0198] For example, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain vanable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRP FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region can comprise an amino acid sequence with at least 1. at least 2. at least 3, at least 4, at least 5. at least 6. at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of the sequences disclosed in Table 1.

[0199] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8. at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid insertions relative to any one of the sequences disclosed in Table 1.

[0200] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2. FR3. CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40. 45. or 50 amino acid insertions relative to any one of the sequences disclosed in Table 1.

[0201] The one or more insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be contiguous, noncontiguous, or a combination thereof.

[0202] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13. at least 14. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20. at least 25. or at least 30 amino acid deletions relative to any one of the sequences disclosed in Table 1.

[0203] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3. CDR3. or FR4 region comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid deletions relative to any one of the sequences disclosed in Table 1.

[0204] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3. CDR3, or FR4 region comprises 1, 2. 3, 4, 5. 6, 7, 8, 9. 10. 11. 12. 13. 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40. 45. or 50 amino acid deletions relative to any one of the sequences disclosed in Table 1 .

[0205] The one or more deletions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, noncontiguous, or a combination thereof.

[0206] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6. at least 7. at least 8, at least 9, at least 10, at least 11, at least 12, at least 13. at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20. at least 25, or at least 30 amino acid substitutions relative to any one of the sequences disclosed in Table 1.

[0207] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises an amino acid sequence with at most 1. at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions relative to any one of the sequences disclosed in Table 1.

[0208] In some embodiments, the extracellular binding protein, a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40. 45. or 50 amino acid substitutions relative to any one of the sequences disclosed in Table 1.

[0209] The one or more substitutions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions can be contiguous, non-contiguous, or a combination thereof. In some embodiments the one or more substitutions are conservative. In some embodiments the one or more substitutions are nonconservative.

[0210] In some cases, the extracellular binding protein comprises a TCR a chain; a TCR P chain; a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRμ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region that is human, humanized, or chimeric.

[0211] In some cases, the KRAS G12 mutant peptide is a KRAS G12V mutant peptide. In some cases, the KRAS G12 mutant peptide comprises the amino acid sequence KLVVVGAVGV. In some cases, the KRAS G12 mutant peptide comprises the amino acid sequence LVVVGAVGV. In some cases, the extracellular binding protein is selective for the KRAS G12V mutant peptide, e.g., specifically, selectively or preferentially binds the KRAS G12V mutant peptide. In some cases, the extracellular binding protein is at least 2-, 3-, 5-, 10-, 25-, 50-, 100-, 200-, 500-, or 1000-, 2000-, 3000-, 4000-, 5000- fold, or 10,000-fold selective for the KRAS G12V mutant peptide versus other 10-mer peptides, for example, a corresponding wild type peptide, or a peptide encoded by a genome of the cell (e.g., that binds to a different KRAS G12-specific TCR, or that is predicted to exhibit off-target binding to the extracellular binding protein).

[0212] In some cases, the extracellular binding protein has a logl 0EC50 for the KRAS G12 mutant peptide of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7. 1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8. 1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9. 1 or less, or about -9.2 or less.

[0213] In some embodiments, a host cell disclosed herein comprises an extracellular binding protein (e.g., TCR) that binds a target antigen of the extracellular binding protein (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex) with an EC50 (e.g., peptide dose at which a half-maximal activation of a T cell population is reached) of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 pM, less than about 100 pM, less than about 50 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, less than about 1 pM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM. less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM. less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, or less than about 100 pM. The host cell can comprise, for example, a modification (e.g., genomic mutation) that results in decreased expression of endogenous TRAC, TRBC1 , and / or TRBC2, a combination thereof. The extracellular binding protein can be a TCR that comprises a Va and VP regions and / or CDRs disclosed herein.

[0214] In some embodiments, a host cell disclosed herein comprises an extracellular binding protein (e.g., TCR) that binds a target antigen of the extracellular binding protein (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex) with an EC50 (e.g., peptide dose at which a half-maximal activation of a T cell population is reached) of at least about 100 mM, at least about 10 mM, at least about 1 mM. at least about 500 pM, at least about 100 pM, at least about 50 pM. at least about 10 pM, at least about 5 pM, at least about 4 pM, at least about 3 pM, at least about 2 pM, at least about 1 pM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, at least about 60 nM, at least about 50 nM, at least about 40 nM, at least about 30 nM, at least about 20 nM, at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 500 pM, at least about 100 pM. The host cell can comprise, for example, a modification (e.g., genomic mutation) that results in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2, a combination thereof. The extracellular binding protein can be a TCR that comprises aVa and VP regions and / or CDRs disclosed herein.

[0215] In some embodiments, an extracellular binding protein (e.g., TCR) binds a target (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex) with a KD of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 pM, less than about 50 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM. less than about 1 pM, less than about 900 nM, less than about 800 nM. less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM. less than about 1 nM, less than about 500 pM. or less than about 100 pM.

[0216] Modifications for reduced expression of endogenous TCR genes

[0217] The host cell can further comprise one or more modifications (e.g., genomic mutation(s)) that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, or a T cell receptor p constant 2 (TRBC2) locus. Decreased expression of TRAC, TRBC1, and / or TRBC2 can, for example, reduce mispairing of transgenic TCR chains that are introduced into a host cell (e.g., anti-KRAS G12V TCRs disclosed herein) with endogenous TCR chains, improve functional expression of the transgenic TCRs, and improve TCR complex signaling and functionality by freeing the available pool of CD3 proteins to bind to the transgenic TCR rather than endogenous TCR chains.

[0218] In some embodiments, the modification can facilitate enhanced in vitro, ex vivo, or in vivo tumor cell killing by engineered immune cells than comparable control cells lacking the modification. In some embodiments, the modification can facilitate enhanced sensitivity to a given (e.g.. low) density of a target antigen (e.g., KRAS G12 mutant peptide) compared to a corresponding control cell lacking the modification.

[0219] In some embodiments, the genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, or a T cell receptor p constant 2 (TRBC2) locus comprises an indel in the TRAC, TRBC1, or TRBC2 locus. In some embodiments, the genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor P constant 1 (TRBC1) locus, or a T cell receptor P constant 2 (TRBC2) locus is a missense mutation and may result in reduced function or stability of a T cell receptor a or T cell receptor p polypeptide encoded in the genome of the host cell. In some embodiments, the genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, or a T cell receptor P constant 2 (TRBC2) locus also results in premature termination of a T cell receptor a or T cell receptor p polypeptide translated from a genomic mRNA of the cell. In some cases, the host cell comprises genomic mutations that cause or contribute to decreased expression of both (i) TRAC; and (ii) TRBC1 or TRBC2. In some cases, the host cell comprises a genomic mutation that causes or contributes to decreased expression of TRAC, TRBC1, and TRBC2. The genomic mutation can be or can comprise an insertion, e.g., of an expression cassette. The genomic mutation can be or can comprise a deletion. The genomic mutation can be or can comprise a substitution.

[0220] In some embodiments, the genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, or a T cell receptor P constant 2 (TRBC2) locus comprises an insertion of a heterologous polynucleotide of the present disclosure (e.g., a polynucleotide comprising a nucleic acid sequence encoding an extracellular binding protein capable of binding a KRAS G12V mutant peptide, or a heterologous polynucleotide encoding an extracellular binding protein of the present disclosure) at the endogenous TRAC. TRBC1, or TRBC2 locus. In some embodiments, the host cell is further modified to decrease and / or knock out expression of endogenous TRAC, TRBC1, and / or TRBC2. Accordingly, in some embodiments, the expression of each of endogenous TRAC, TRBC1, and TRBC2 is decreased and / or knocked out. In some embodiments, the modification comprises deletion of, for example, at least 10%. at least 20%. at least 30%. at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the open reading frame of endogenous TRAC, TRBC1 , or TRBC2.

[0221] In some embodiments, the modification comprises knockdow n of expression of the TRAC, TRBC1, or TRBC2, for example, using a shRNA or siRNA. In some embodiments, the modification comprises a genomic disruption. In some embodiments, the modification comprises insertion of, for example, a transposon or a premature stop codon.

[0222] In some embodiments, expression of endogenous TRAC, TRBC1, and / or TRBC2 is reduced by at least 5%, 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%, 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold. In some embodiments, expression of TRAC, TRBC1, and / or TRBC2 is eliminated or substantially eliminated. In some embodiments, expression of TRAC, TRBC1, and / or TRBC2 is reduced to below a limit of detection. The reduced expression of TRAC, TRBC1, and / or TRBC2 can be determined, for example, by a flow cytometric assay (e.g.. for proportion of positive cells or mean fluorescence intensity, in a population of interest).

[0223] In some embodiments, the reduction in expression of endogenous TRAC, TRBC1, and / or TRBC2 is found in at least 5%, 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%, at least 95%, at least 97%, or at least 99% of host cells in a population. In some cases, the reduction of expression is found by a genomic sequencing method.

[0224] In some embodiments, a population of host cells disclosed herein comprising one or more modifications (e.g., genomic mutation(s)) that result in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibits at least 5%, 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%, 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased killing of target cells as compared to a population of control cells (for example, cells without reduced expression of TRAC, TRBC1, and / or TRBC2). The killing of target cells can be, for example, as determined by an in vitro cytotoxicity assay. The host cells can comprise an extracellular binding protein (e.g.. a TCR comprising Va and VP regions and / or CDRs disclosed herein) that binds a target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex).

[0225] In some embodiments, a population of host cells disclosed herein comprising one or more modifications (e.g., genomic mutation(s)) that result in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibits at least 5%, 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%, 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased activation in response to target cells as compared to a population of control cells (for example, cells without reduced expression of TRAC, TRBC1, and / or TRBC2). The host cells can comprise an extracellular binding protein (e.g., a TCR comprising Va and VP regions and / or CDRs disclosed herein) that binds a target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex). The activation can be, for example, as determined by an assay for determining expression an activation marker (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon) upon exposure to target cells that express or present the target antigen.

[0226] In some embodiments, a population of host cells disclosed herein comprising one or more modifications (e.g., genomic mutation(s)) that result in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibits at least 5%, 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%, 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased avidity for a target antigen of an extracellular binding protein as compared to a population of control cells (for example, cells without reduced expression of TRAC, TRBC1, and / or TRBC2). The host cells can comprise an extracellular binding protein (e.g., a TCR comprising Va and VP regions and / or CDRs disclosed herein) that binds a target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex). The increase in avidity can be. for example, as determined by an assay for determining expression an activation marker (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon) upon exposure to target cells that express or present the target antigen, or and / or an assay to determine EC50 (e.g., peptide dose at which a half- maximal activation of a T cell population is reached).

[0227] In some embodiments, a population of host cells disclosed herein comprising one or more modifications (e.g., genomic mutation(s)) that result in decreased expression of endogenous TRAC, TRBC1. and / or TRBC2 exhibits at least 5%, 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%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, or at least 50 fold increased binding to a target antigen of an extracellular binding protein as compared to a population of control cells (for example, cells without reduced expression of TRAC, TRBC1, and / or TRBC2). The host cells can comprise an extracellular binding protein (e.g.. a TCR comprising Va and VP regions and / or CDRs disclosed herein) that binds the target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex). The increase in binding can be, for example, as determined by an assay comprising staining with peptide-HLA multimers (e.g., tetramers or pentamers).

[0228] In some embodiments, a population of host cells disclosed herein comprising one or more modifications (e.g., genomic mutation(s)) that result in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2 exhibits at least 5%, 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%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100-fold, or at least 500-fold increased expression (e g., cell surface expression) of an extracellular binding protein as compared to a population of control cells (for example, cells without reduced expression of TRAC. TRBC1, and / or TRBC2). The extracellular binding protein can be a TCR comprising an alpha chain and beta chain (e.g., with Va and V0 regions and / or CDRs disclosed herein) that binds the target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex). The increase in expression can be, for example, as determined by an assay comprising staining with peptide-HLA multimers (e.g., tetramers or pentamers) specific for the extracellular binding protein.

[0229] In some embodiments, the population of host cells comprises decreased expression of endogenous TRAC relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRBC1 relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRBC2 relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRAC and TRBC1 relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRAC and TRBC2 relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRBC1 and TRBC2 relative to the control cells. In some embodiments, the population of host cells comprises decreased expression of endogenous TRAC, TRBC1, and TRBC2 relative to the control cells.

[0230] Additional polypeptides

[0231] A host cell can be engineered to comprise further modifications in addition to an extracellular binding protein and a modification that causes or contributes to decreased expression of TRAC, TRBC 1 , and / or TRBC2.

[0232] A host cell can comprise a transgenic polynucleotide encoding a polypeptide that comprises a CD8 co-receptor a (CD8a) chain or an extracellular portion thereof, and / or a transgenic polynucleotide encoding a CD8 co-receptor 0 (CD80) chain polypeptide or an extracellular portion thereof. A host cell can be engineered to express a CD8 co-receptor disclosed herein, e.g., a CD8a chain and / or a CD80 chain. Illustrative, non-limiting examples of CD8a and CD80 amino acid sequences that can be used include those provided below, and variants thereof.

[0233] A host cell can comprise a transgenic polynucleotide encoding a Fas-41BB fusion protein. An illustrative, non-limiting example of a Fas 41-BB fusion protein amino acid sequence that can be used includes the sequence provided below, and variants thereof.

[0234] The Fas-41BB fusion protein can comprise, for example, an extracellular domain of Fas or a FasL-binding fragment thereof, and an intracellular signaling domain of 4 IBB or a signaling domain thereof. A Fas-41BB fusion protein can be useful for. for example, converting a signal initiated by the binding of Fas to its target (e.g., FasL) into a positive (e.g., costimulatory) signal generated by the 4- IBB intracellular signaling domain, thereby improving anti-cancer immune functionality of a host cell disclosed herein (e.g., increased proliferation, survival in the tumor microenvironment, and metabolism to support T cell activation and memory development). The extracellular component can comprise all or a portion of the extracellular domain of Fas, or can be truncated to maintain a short spatial distance between the host cell and an interaction partner (e.g., ~9aas) upon receptor-ligand interaction. The Fas-41BB fusion protein can comprise a transmembrane domain, for example, a Fas, 4-1BB, or CD28 transmembrane domain.

[0235] A host cell can comprise a transgenic polynucleotide encoding a chimeric fusion protein that comprises an IL7R intracellular signaling domain. The chimeric fusion protein can comprise, for example, an intracellular portion of an Interleukin 7 Receptor A (IL7RA) polypeptide, or a portion or variant thereof that is capable of contributing to an IL-7 signal in a host cell. A chimeric IL7R fusion protein can. for example, provide a “signal 3?’ to increase STAT5 phosphorylation and host cell functionality, enhance proliferation of a host cell, increase host cell survival (e g., in the tumor microenvironment), and / or enhance chemokine receptor expression.

[0236] Interleukin-7 receptor subunit alpha can also be referred to as IL7R-a, as IL7RA, as IL-7R-alpha, as ILRA, as Interleukin-7 receptor-a, as interleukin 7 receptor, as Cluster of Differentiation 127 as CD127, or as CDW127. Illustrative, non-limiting examples of IL7RA intracellular domain and transmembrane domain amino acid sequences that can be used include those provided below, and variants thereof.

[0237] In some embodiments, the mutation enables or facilitates homodimerization of the receptor. In some embodiments, the mutation comprises an insertion of a trimer peptide of cysteine, proline, threonine (CPT) into the transmembrane domain. In some embodiments, the threonine of the CPT insertion is not threonine but another amino acid, and in at least specific cases that other amino acid is or is not cysteine or proline.

[0238] In some embodiments, the chimeric fusion protein comprises a transmembrane domain of IL7R, IL2RA, IL2RB, IL2RG, IL14R, IL15R, IL9R, IL21R, CD2, CD40L, CD58, CD80, or SIRPa.

[0239] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising: (i) an extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof that is capable of binding a CD28 or CTLA-4 polypeptide; (ii) an extracellular domain of a Cluster of Differentiation 58 (CD58) polypeptide, or a portion or variant thereof that is capable of binding a Cluster of Differentiation 2 (CD2) polypepride; (iii) an extracellular domain of a Signal Regulatory Protein Alpha (SIRPa) polypeptide, or a portion or variant thereof that is capable of binding a Cluster of Differentiation 47 (CD47) polypeptide; (iv) an extracellular domain of a Cluster of Differentiation 40L (CD40L) polypeptide, or a portion or variant thereof that is capable of binding a CD40 polypeptide; (v) an extracellular domain of a Cluster of Differentiation 2 (CD2) receptor, or a portion or variant thereof that is capable of binding a CD58 polypeptide; or (vi) an extracellular domain of a Cluster of Differentiation 34 (CD34) polypeptide.

[0240] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising an extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof that is capable of binding a CD28 or CTLA-4 polypeptide.

[0241] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits at least 5%, 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%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 50 fold, or at least 100 fold, at least 500 fold, or at least 1000 fold increased proliferation in response to target cells (e.g., that present a KRAS G12V peptide) as compared to a population of control cells (for example, corresponding cells lacking the Fas-41BB fusion protein or chimeric IL7R polypeptide). The proliferation can be, for example, as determined by an in vitro lymphoproliferation assay or measurement of host cell numbers after co-incubation. The host cells can comprise an extracellular binding protein (e.g., a TCR comprising Va and VP regions and / or CDRs disclosed herein), and / or a modification that results in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2.

[0242] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits at least 5%, 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%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 50 fold, or at least 100 fold, at least 500 fold, or at least 1000 fold increased killing of target cells as compared to a population of control cells (for example, corresponding cells lacking the Fas-41BB fusion protein or chimeric IL7R polypeptide). The killing of target cells can be, for example, as determined by an in vitro cytotoxicity assay. The host cells can comprise an extracellular binding protein (e.g., a TCR comprising Va and VP regions and / or CDRs disclosed herein), and / or a modification that results in decreased expression of endogenous TRAC, TRBC1, and / or TRBC2.

[0243] A nucleic acid encoding a polypeptide disclosed herein (e g., extracellular binding protein, CD8 co-receptor chain or an extracellular portion thereof, Fas-41BB fusion protein, or chimeric IL7R fusion protein) can encode a signal peptide. In some cases, a polypeptide of the disclosure comprises a signal peptide. A signal peptide can be cleaved off during processing of the polypeptide, thus in some cases a mature polypeptide disclosed herein does not contain a signal peptide.

[0244] A signal peptide at the N-terminus of a protein can be involved in transport of the protein to or through a membrane, transport to different a membranous cellular compartment, or secretion of the protein from the cell. A nucleic acid encoding a protein of the disclosure can encode a signal peptide to facilitate membrane insertion and surface localization of the protein. A signal peptide can be selected for its ability’ to facilitate ER processing and cell surface localization of the protein. Any suitable signal peptide can be used. In some cases, the signal peptide can comprise a G-CSF signal peptide or a CD8a signal peptide. A signal peptide can be about 10 to about 40 amino acids in length. In some cases, a signal peptide is at least about 10, 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or more. In some cases, a signal peptide is at most about 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or less. In some cases, a signal peptide is about 16-30 amino acids in length.

[0245] Compositions

[0246] In some cases, the present disclosure provides for a pharmaceutically acceptable composition comprising a plurality of host cells described herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some cases, the composition comprises a CD4+ T cell population and / or a CD8+ T cell population bearing: (i) the extracellular binding protein; and (ii) one or more genomic mutations that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, a T cell receptor P constant 2 (TRBC2) locus, or a combination thereof. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition comprises a CD8+ cell population comprising (ii) at least about 20%, at least about 30%. at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells.

[0247] In some embodiments, the CD4+ and / or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a (CD8a) chain or a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P (CD8P) chain. In some embodiments, the CD4+ and / or CD8+ cells express a CD8 co-receptor disclosed herein, e.g., a CD8a chain and / or a CD80 chain.

[0248] In some cases, the composition comprises a CD4+ T cell population and / or a CD8+ T cell population bearing: (i) the extracellular binding protein; and (ii) one or more genomic mutations that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor 0 constant 1 (TRBC1) locus, a T cell receptor 0 constant 2 (TRBC2) locus, or a combination thereof. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%. or at least about 95% modified CD4+ T cells. In some embodiments, the composition comprises a CD8+ cell population comprising (ii) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells. In some embodiments, the CD4+ or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a (CD8a) chain or a resulting polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor 0 (CD80) chain. In some cases, the composition comprises both CD4+ and CD8+ cells bearing: (i) the extracellular binding protein and (ii) a genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor 0 constant 1 (TRBC1) locus, or a T cell receptor 0 constant 2 (TRBC2) locus. In some cases, the composition comprises both of (ii) a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a (CD8a) chain or a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor 0 (CD80) chain.

[0249] In some cases, the composition comprises both CD4+ and CD8+ cells bearing: (i) the extracellular binding protein; and either or both of (ii) a genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor 0 constant 1 (TRBC1) locus, or a T cell receptor 0 constant 2 (TRBC2) locus. In some embodiments, the CD4+ or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a (CD8a) chain or a resulting polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor 0 (CD80) chain. In some embodiments, the CD4+ or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a (CD8a) chain or a resulting polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor P (CD80) chain. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises a CD8+ cell population comprising (ii) at least about 30%. at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells. In some cases, the composition comprises a reduced amount or substantially no naive T cells. In some embodiments, the composition comprises about a 1 : 1 ratio of CD4+ to CD8+ T cells. In some cases, the composition comprises both CD4+ and CD8+ cells bearing: (i) the extracellular binding protein: and either or both of (ii) a genomic mutation that causes or contributes to decreased expression of an endogenous T cell receptor a constant (TRAC), a T cell receptor p constant 1 (TRBC1) locus, or a T cell receptor p constant 2 (TRBC2) locus. In some embodiments, the CD4+ or CD8+ cells further comprise a transgenic polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a (CD8a) chain or a resulting polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P (CD8P) chain. In some embodiments, the composition comprises a CD4+ cell population comprising (i) at least about 30%, at least about 40%. at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises a CD8+ cell population comprising at least about 30%. at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%. at least about 90%. or at least about 95% modified CD8+ T cells. In some cases, the composition comprises a reduced amount or substantially no naive T cells. In some embodiments, the composition comprises about a 1: 1 ratio, about a 1:2 ratio, about a 1:3 ratio, about a 1:4 ratio, about a 1:5 ratio, about a 1:6 ratio, about a 1:7 ratio, about a 1 : 8 ratio, about a 1 : 9 ratio, about a 1 : 10 ratio, about a 2 : 1 ratio, about a 3 : 1 ratio, about a 4: 1 ratio, about a 5: 1 ratio, about a 6: 1 ratio, about a 7: 1 ratio, about an 8: 1 ratio, about a 9: 1 ratio, or about a 10: 1 ratio of CD4+ to CD8+ T cells.

[0250] In some cases, the carrier or excipient comprises albumin. In some cases, the diluent comprises physiologically normal saline. Suitable excipients can also include water, saline, dextrose, glycerol, or the like, and combinations thereof. In some embodiments, a composition comprises a suitable infusion media. Suitable infusion media can be any isotonic medium formulation, normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, or Ringer's lactate can be utilized. An infusion medium can be supplemented with human serum albumin or other human serum components.

[0251] Polynucleotides or vectors

[0252] In some aspects, the present disclosure provides for a polynucleotide comprising an open reading frame encoding an extracellular binding protein (e.g., an extracellular binding protein capable of binding a KRAS G12V mutant peptide). The open reading frame may be operatively linked to a promoter (e.g., heterogenous promoter). The extracellular binding protein may comprise a TCR a chain variable (Va) domain; a TCR P chain variable (VP) domain; a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; or a TCRP FR1. CDR1, FR2, CDR2, FR3, CDR3, or FR4 region disclosed herein, for example, comprising a sequence having at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any of the sequences described herein. The polynucleotide may be codon optimized.

[0253] In some cases, the heterogenous promoter is not a mammalian promoter. In some cases, the heterogenous promoter is a constitutive promoter that is not a TCR promoter. In some cases, the heterogenous promoter is a viral promoter. In some cases, the heterogenous promoter is a mammalian promoter. In some cases, the heterogenous promoter is a human promoter. In some cases, the heterogenous promoter is a synthetic promoter. In some cases, the heterogenous promoter is an inducible promoter. In some cases, the heterogenous promoter is a tissue-specific promoter. In some cases, the heterogenous promoter is an immune cell-specific promoter.

[0254] In some embodiments, the promoter is a murine stem cell virus (MSCV) promoter. In some embodiments, the polynucleotide is promoterless. In some embodiments, the promoterless polynucleotide is designed to be operatively linked to an endogenous promoter at the site of insertion (e.g., at the TRAC locus). In some embodiments, the promoter is an elongation factor-1 alpha (EF-la) promoter. In some embodiments, the promoter has at least about 85% sequence identity7to the following exemplary7sequence: EF-la promoter

[0255] In some embodiments, the promoter is a Mei otic Nuclear Divisions 1 (MND1) promoter.

[0256] In some embodiments, the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises a KRAS G12V mutant peptide. In some cases, the KRAS G12V mutant peptide comprises the amino acid sequence KLVVVGAVGV. In some embodiments, the extracellular binding protein is human, humanized, or chimeric. In some embodiments, the extracellular binding protein is selective for the KRAS G12V mutant peptide. In some embodiments, the extracellular binding protein has a logI0EC50 for the KRAS G12V mutant peptide of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -

[0257] 6.8 or less, about -6.9 or less, about -7.0 or less, about -7. 1 or less, about -7.2 or less, about - 7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -

[0258] 7.8 or less, about -7.9 or less, about -8.0 or less, about -8. 1 or less, about -8.2 or less, about - 8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about - 8.8 or less, about -8.9 or less, about -9 or less, about -9. 1 or less, or about -9.2 or less.

[0259] In some aspects, the present disclosure provides for a vector comprising any of the polynucleotides described herein. In some cases, the vector is a viral vector, such as a lentiviral vector, a / -retroviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the vector is a non-viral vector, for example, a plasmid, nanoplasmid, minicircle, a midge, a MIP, or a doggybone, a lipid-based nanoparticle, a liposome, a circular polynucleotide (e.g., DNA or RNA), a linear polynucleotide (e.g., a DNA or RNA), or a combination thereof.

[0260] Methods of treatment

[0261] In some aspects, the present disclosure provides for a method of treating a disease or disorder associated with a KRAS G12V mutation in a subject, comprising administering to the subject an effective amount of any of the host cells or compositions described herein. In some embodiments, the host cell is autologous to the subject. In some embodiments, the host cell is allogenic to the subject. In some embodiments, the host cell is HLA-matched to the subject, for example, HL A matched at all typed HLA alleles. In some embodiments, a host cell and a subject can be HLA-typed HLA- A, HLA-B, HLA-C, and / or HLA-DR alleles. In some embodiments, the host cell and subject are matched for at least 1, at least 2, at least 3, at least 4. at least 5. at least 6, at least 7, or at least 8 HLA alleles. In some embodiments, the host cell is haploidentical to the subject.

[0262] In some embodiments, the subject is positive for an HLA-A*02 allele. In some embodiments, the subject is positive for an HLA-A*02:01 allele.

[0263] In some embodiments, the disease or disorder comprises a cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma: Chronic Myelomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatosis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary7AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; or thyroid gland papillary^ carcinoma.

[0264] In some embodiments, the method further comprises genoty ping a tumor of the subject for a KRAS G12V allele prior to the administering. In some embodiments, the method further comprises genotyping the subject for an HLA-A allele prior to the administering. In some cases, the subject is determined to carry' a KRAS G12V allele prior to the administering. In some cases, the subject has been genotyped for an HLA-A allele prior to the administering.

[0265] An effective amount of a pharmaceutical composition can describe an amount sufficient, at dosages and for periods of time needed, to achieve the predetermined clinical results or beneficial treatment. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount" may be used in reference to treatment, whereas "prophylactically effective amount" may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or diseasestate (e.g., recurrence) as a preventative course.

[0266] Administration may be affected continuously or intermittently, and parenterally. A composition can be administered locally (e.g.. intratumorally) or systemically (e.g., intravenously). Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous or sequential delivery of multiple agents in any order and on any dosing schedule. Methods disclosed herein may further include administering one or more additional agents to treat the disease or disorder in a combination therapy. For example, in certain embodiments, a combination therapy comprises administering an engineered host cell with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, a combination therapy comprises administering a host cell with an agonist of a stimulatory immune checkpoint agent. In some embodiments, a combination therapy comprises administering a host cell with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.

[0267] Methods of manufacturing

[0268] In some aspects, the present disclosure provides for a method of manufactunng a host cell, comprising contacting to the host cell: (a) any of the polynucleotides or vectors described herein. In some cases, the method further comprises contacting to the host cell: (b) an endonuclease (e.g., Cas endonuclease, such as a class II, ty pe V Cas endonuclease); and (c) a guide RNA compatible with the endonuclease (e.g., compatible with the class II, type V Cas endonuclease), wherein the guide RNA is configured to hybridize to an endogenous T cell receptor constant region locus of the host cell prior to the polynucleotide or vector or after the polynucleotide or vector. In some embodiments, the contacting comprises transfection or transduction. In some embodiments, the transfection comprises electroporation. In some embodiments, the T cell receptor constant region locus is a TRAC. TRBC1, or TRBC2 locus.

[0269] In some embodiments, the host cell comprises an immune cell or a precursor thereof. In some embodiments, the immune cell comprises a T cell, aNK cell, aNK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In some embodiments, the immune cell comprises a T cell, wherein the T cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y5 T cell, or any combination thereof.

[0270] Cells can be engineered to comprise or be capable of expressing an extracellular binding protein, an additional polypeptide disclosed herein, and / or to reduce expression of an endogenous TCR gene. For example, cell engineering techniques disclosed herein and / or known to a skilled person can be used to modify cells to comprise a recombinant nucleic acid that encodes an extracellular binding protein of the disclosure, and / or to introduce a modification to reduce expression of endogenous TRAC, TRBC1 and / or TRBC2, thereby generating host cells (such as engineered T cells). The methods can comprise contacting a cell with a recombinant nucleic acid, or with a vector that comprises the recombinant nucleic acid, under conditions that permit uptake of the recombinant nucleic acid by the cell. A recombinant nucleic acid can comprise a nucleotide sequence that encodes an extracellular binding protein disclosed herein or a component thereof. In some cases, a recombinant nucleic acid is utilized to alter a genome of a cell.

[0271] A recombinant nucleic can be a substance whose molecules comprise or consist essentially of nucleotides linked in a chain. Non-limiting examples of the recombinant nucleic include a circular nucleic acid, a DNA, a single stranded DNA, a double stranded DNA, a genomic DNA, a plasmid, a nanoplasmid, a plasmid DNA, a viral DNA. a mini circle (e.g.. lacking a bacterial origin of replication), and an RNA.

[0272] A recombinant nucleic acid can include one or more homology arms, for example, comprising sequences that are complementary' to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination). A recombinant nucleic acid can comprise one or more promoter regions, barcodes, restriction sites, cleavage sites, endonuclease recognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites may be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance properties (e.g.. antibiotic resistance for selection), or any combination thereof. A recombinant nucleic acid may also include transcriptional or translational regulatory sequences, for example, one or more promoters, enhancers, insulators, internal ribosome entry' sites, sequences encoding 2A linkers and / or polyadenylation signals.

[0273] Recombinant nucleic acids can be assembled by a variety of methods, e.g., by automated solid-phase synthesis. A recombinant nucleic acid can be constructed using standard solid-phase DNA / RNA synthesis. A recombinant nucleic acid can also be constructed using a synthetic procedure. A recombinant nucleic acid can be synthesized manually or in a fully automated fashion. In some cases, a synthetic procedure may comprise 5'-hydroxyl oligonucleotides that can be initially transformed into corresponding 5’-H- phosphonate mono esters, subsequently oxidized in the presence of imidazole to activated 5'- phosphorimidazolidates, and finally reacted with pyrophosphate on a solid support. This procedure may include a purification step after the synthesis such as PAGE, HPLC, MS, or any combination thereof. Recombinant nucleic acids can be purchased commercially. Recombinant nucleic acid described herein can be modified. In some cases, a recombinant nucleic acid can be modified to make it less immunogenic and more stable for transfection into a cell.

[0274] For targeted integration, a recombinant nucleic acid sequence to be inserted can be flanked by homolog}' arms comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination and / or homology- directed repair, HDR). A double stranded break can be introduced at a target site in the genome, and the homology arms can promote insertion of the recombinant nucleic acid. In some cases, a recombinant nucleic acid can be excised from a vector, such as a nanoplasmid (e.g., via a nuclease), and inserted into the genome of the cell.

[0275] A recombinant nucleic acid can be inserted in a safe harbor locus. A safe harbor can comprise a genomic location where a recombinant nucleic acid can integrate and function without substantially perturbing endogenous activity7, for example, with a relatively low impact on local or global gene expression. For example, one or more recombinant nucleic acids can be inserted into any one of HPRT. an AAVS site (E.G.. AAVS1, AAVS2, etc.), CCR5, hROSA26, and / or any combination thereof. A recombinant nucleic acid can be inserted in an intergenic region. A recombinant nucleic acid can be inserted in a non-coding region. A recombinant nucleic acid can be inserted within a gene. In some cases, a recombinant nucleic acid can disrupt a gene it is inserted into (e.g., reduce or eliminate expression of the disrupted gene). A disrupted gene can be for example, an endogenous TCR gene (e g., TRAC, TCRB, TCRBC1, TRBC2, TRG, TRD), or an immune checkpoint gene (e.g., PD-1, CTLA-4). A recombinant nucleic acid can be inserted adjacent to or near to an endogenous promoter such that the recombinant nucleic acid is operably linked to the endogenous promoter.

[0276] A variety of enzymes can catalyze generation of a double-stranded break in the genome and / or insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR systems, CRISPR-associated polypeptide (Cas), TALEN, zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptide, meganuclease, Mega-TAL, transposon-based systems, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0277] A CRISPR system can be utilized to facilitate insertion of a recombinant nucleic acid encoding an extracellular binding protein or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome or a random site of a genome.

[0278] In some cases, a CRISPR system comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR- associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, or type VI CRISPR-associated (Cas) polypeptides a derivative, variant, or functional fragment thereof.

[0279] In some embodiments, a CRISPR system comprises a Class I system or endonuclease (e.g., Type I, Type III or Type IV Cas proteins). A class I system can be of the I-A, I-B, I-C, I-U, I-D, I-E. I-F, IV-A, IV-B, III-A, III-D, III-C, or III-B subtype.

[0280] In some embodiments, a CRISPR system comprises a Class II system or endonuclease (e g., Type II, Type V, or Type VI). A class II, Type II system can be of the II-A, II-B, II-C1, or II-C2 subtype. A class II, Type V systems can of the V-A, V-Bl, V-B2, V-C, V-D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H. V-I, V-K (V-U5), V-Ul, V-U2, or V-U4 subtype. A Class II, Type IV systems can be of the: VI- A, VI-B1, VI-B2. VI-C, or VI-D subtype.

[0281] In some embodiments, a Cas protein used in a method disclosed herein is a class II endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V Cas endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II. type V-A Cas endonuclease.

[0282] Non-limiting examples of Cas proteins that can be used in the CRISPR systems include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6. Cmrl, Cmr3. Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl O, Csxl 6, CsaX, Csx3, Csxl , Csxl S, Csfl , Csf2, CsO, Csf4, Cpfl, c2cl , c2c3, Cas9HiFi, homologues thereof, and modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A Cas protein can be a high-fidelity Cas protein. Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpfl family that display cleavage activity in mammalian cells. In some embodiments, a gene editing system comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the gene editing moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein. In some embodiments, the gRNA comprises a targeting segment which exhibits at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to a target polynucleotide. Multiple gRNAs can be used, e.g., to simultaneously or sequentially target TRAC, TRBC 1, and / or TRBC2.

[0283] In some cases, a dual nickase approach may be used to introduce a double stranded break. Cas proteins can be mutated at certain amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break. A nickase along with two distinct guide RNAs targeting opposite strands may be utilized to generate a DSB within a target site (often referred to as a “double nick” or “dual nickase” CRISPR system).

[0284] A transposon-based system can be utilized for insertion of a recombinant nucleic acid encoding an extracellular binding protein of the disclosure or a component thereof into a genome, or for disruption of a TCR encoding gene. A transposon can comprise a recombinant nucleic acid that can be inserted into a DNA sequence. A class I transposon can be transcribed into an RNA intermediate, then reverse transcribed and inserted into a DNA sequence. A class II transposon can comprise a DNA sequence that is excised from one DNA sequence and / or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector that contains a sequence (e.g., comprising a transgene) flanked by inverted terminal repeats, and (ii) a source for the transposase enzyme. A transposon system (e.g., class II transposon system) can direct the integration of a recombinant nucleic acid sequence encoding an extracellular binding protein or a component thereof, while leaving behind the rest of the vector. A transposon and a transposase can be introduced into a cell. In some cases, a vector that encodes a transposase and comprises a recombinant nucleic acid is introduced into a cell, and the transposase is expressed and mediates insertion of the transposon into the genome.

[0285] Examples of transposon-based systems that can be used include, but are not limited to, sleeping beauty' (e.g., derived from the genome of salmonid fish); piggy back (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g.. derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON. In some embodiments an extracellular binding protein or other polypeptide can be expressed in an host cell without genomic integration of a recombinant nucleic acid that encodes the extracellular binding protein or other polypeptide. For example, an extracellular binding protein or other polypeptide can be expressed from an episomal vector, such as a DNA, RNA, circular DNA, circular RNA, minicircle, and the like. An extracellular binding protein or other polypeptide can be transiently expressed. For example, expression of an extracellular binding protein or other polypeptide can be reduced as a nucleic acid that encodes it is degraded. One method of generating host cells is through the use of a ribonucleic acid (RNA) system, e.g., a system that involves delivering one or more recombinant nucleic acids as an RNA. In some cases, the use of RNA can minimize DNA- induced toxicity and immunogenicity sometimes observed with the use of DNA.

[0286] In some cases, one or more recombinant nucleic acids of the disclosure can be inserted randomly into the genome of a cell. For instance, a recombinant nucleic acid can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively or additionally, a recombinant nucleic acid can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a noncoding region.

[0287] One or more recombinant nucleic acids and / or gene editing components can be delivered to a cell by any suitable method, for example, using any suitable vector. A vector can be or can comprise a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, a transposon, and the like. Any vector systems can be used including, but not limited to, DNA vectors, RNA vectors, ribonucleoprotein vectors, hybrid DNA-RNA vectors, plasmid vectors, nanoplasmid vectors, minicircle vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery' vehicle such as a liposome, lipid nanoparticle, or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. In some cases, one vector is used. In some cases, two vectors are used. In some cases, three or more vectors are used.

[0288] In some cases, recombinant nucleic acids and / or gene editing components of the disclosure can be delivered to cells without the use of vectors. In some cases, one or more recombinant nucleic acids and / or gene editing components of the disclosure can be delivered to cells via vectors, and one or more recombinant nucleic acids and / or gene editing components can be delivered without the use of vectors.

[0289] Cells can be genetically engineered to comprise a recombinant nucleic acid that encodes an extracellular binding protein and / or modification to reduce expression of TRAC, TRB, TRBC1, and / or TRBC2 ex vivo. For example, cells can be taken from a subject in one or more blood draws and / or apheresis procedures, modified ex vivo, optionally selected and / or expanded before and / or after genetic modification, and optionally re-introduced into the subject or a different subject by infusion or injection.

[0290] In some cases, cells are genetically engineered to comprise an extracellular binding protein of the disclosure and / or modification to reduce expression of endogenous TRAC, TRB. TRBC1. and / or TRBC2 in vivo. For example, a vector can be used to deliver gene editing components to cells in a subject without removing the cells from the subject. Vectors can be delivered in vivo by administration to an individual subject, for example, by parenteral administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application.

[0291] Methods to introduce gene editing components into a cell include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including adenoviral, AAV, and retroviral vectors.

[0292] Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the Xenon Electroporation System (ThermoFisher Scientific), or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery7of nucleic acids into a cell. Electroporation parameters may be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and / or cell viability.

[0293] Cells can be selected or enriched for having or not having one or more given factors (e.g.. cells may be separated based on the presence or absence of one or more factors). Selection techniques include positive selection and negative selection techniques, e.g., fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS). In some cases, cells can be selected before gene editing, for example, to enrich for a population of cells disclosed herein (e.g., immune cells, such as T cells or a T cell subset disclosed herein, such as gamma delta T cells or alpha beta T cells). Cells can be selected after gene editing, for example, to enrich for a population of cells disclosed herein (e.g., host cells that express an extracellular binding protein or additional polypeptide, and / or comprise a modification to reduce expression of endogenous TRAC, TRB, TRBC1, and / or TRBC2). Host cells can be selected or enriched based on a tag or marker, such as an epitope tag. The tag or marker can be appended to the extracellular binding protein. In some embodiments, the tag or marker is not appended to the extracellular binding protein. The tag or marker can be co-expressed with the extracellular binding protein as disclosed herein. The tag or marker can comprise a reporter gene, such as a fluorescent protein.

[0294] Cells can be selected, enriched, or expanded on the basis of being positive or negative for a given factor. In some embodiments, cells are selected, enriched, or expanded on the basis of being positive for two or more factors. In some embodiments, cells can be selected, enriched, or expanded on the basis of being positive for one or more factors, and negative for one or more factors.

[0295] In some cases, a selectable marker is introduced to a cell, e.g.. together with or as part of a recombinant nucleic acid encoding an extracellular binding protein, so that cells that comprise the extracellular binding protein or modification express the selectable marker and can be selected, enriched, or expanded. In some cases, a selectable marker is an antibiotic resistance gene, and cells that do not express the antibiotic resistance gene can be killed by treatment with the antibiotic (e.g., to select or enrich for cells that comprise an extracellular binding protein). In some embodiments, the selectable marker is an epitope tag.

[0296] Expression of an extracellular binding protein, TRAC, TRB, TRBC 1, and / or TRBC2 of the disclosure can be quantified, for example, by qPCR, RNA sequencing, western blot, or flow cytometry.

[0297] In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a recombinant nucleic acid, after gene editing or delivery of a recombinant nucleic acid, before selection, after selection, before expansion, after expansion, or a combination thereof. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a recombinant nucleic acid. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after gene editing or delivery of a recombinant nucleic acid. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before expansion. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after expansion.

[0298] Methods of Characterizing a Disease or Disorder

[0299] The instant disclosure also provides methods of characterizing a disease or disorder described herein (e.g., a neoplasia). In some embodiments, a subject, a disease or disorder of the subject, or a biological sample of the subject is characterized prior to treatment or administration of an agent of the disclosure.

[0300] In some embodiments, the subject, a disease or disorder (e.g.. a neoplasia) of the subject, or a biological sample of the subject is characterized as comprising a KRAS mutation (e.g., KRAS G12V mutation) prior to treatment of the subject. Characterization of KRAS mutants in a subject is well known in the art and described, for example, in Shackelford et al., 2012, Genes & Cancer 3(7-8):459-66.

[0301] Kits

[0302] The instant disclosure also provides kits containing agents of this disclosure for use in the methods of the present disclosure. Kits of the instant disclosure may include one or more containers comprising an agent for treatment of a neoplasia. In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, these instructions comprise a description of use of the agent to treat, e.g., a neoplasia, according to any of the methods of this disclosure.

[0303] Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for practicing any of the methods described herein.

[0304] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology ” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0305] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0306] EXAMPLES

[0307] Example 1: Identification and Characterization of TCRs

[0308] T cell receptors (TCRs) were isolated from circulating T cells in healthy donors having the human leukocyte antigen (HLA) HLA-A*02 serotype. These TCRs were narrowed dow n to two batches of TCRs which recognize Kirsten rat sarcoma viral oncogene homologue (KRAS) G12V mutant peptides in an HLA-A*02 context. Batch 1 was evaluated with both mouse and human constant regions for the TCR, and Batch 2 was only evaluated with human constant regions for the TCR. Each batch was then transduced into T cells using a lentiviral vector which included a polynucleotide encoding TCRa / p chains from a TCR of interest, and CD8a / p polypeptides, each separated with 2A self-cleaving peptides. Transduction efficiency of each batch of TCRs was then analyzed (FIG. 1). Both batches of TCRs showed high transduction efficiency. The sequences of the TCRs are shown in Table 1 Table 1: TCRs Recognizing KRAS G12V Peptide in HLA-A*02 Context

[0309] TCRs from Batch 1 (FIG. 2) and Batch 2 (FIG. 6) were then tested against antigenic tetramers for antigen recognition. The tetramer was made from four subunits, each subunit including a HL A- A* 02: KRAS G12V peptide complex. TCRs from both batches showed recognition for the tetramer.

[0310] Batches 1 and 2 were tested in cell killing / cytotoxicity assays in multiple tumor models displaying the KRAS G12V peptide and selected TCRs were chosen for further analysis. T cells transduced with Batch 1 TCRs were introduced to pancreatic adenocarcinoma cell models (CFPAC1, DANG. PATU8902) and colorectal adenocarcinoma cell models (SW480. SW527, SK-CO1) expressing KRAS G12V peptide and endogenous HLA-A*02 (FIG. 4). T cells transduced with Batch 2 TCRs were introduced to pancreatic adenocarcinoma cell models (CFPAC1, DANG) and colorectal adenocarcinoma cell models (SW527, SK-CO1) expressing KRAS G12V peptide and endogenous HLA-A*02 (FIG. 8). Tumor cell confluence was measured. The cytotoxicity assay identified TCR 130 from Batch 1 and TCRs 240 and 243 from Batch 2 as the TCRs chosen for further analysis.

[0311] The TCRs from Batch 1 (TCR 130) and Batch 2 (TCRs 240 and 243) were next tested for sensitivity for the KRAS G12V peptide. T cells from 2 different donors were transduced with the TCRs from Batch 1 (TCR 130) and Batch 2 (TCRs 240 and 243). The transduced TCR T cells were co-cultured with T2 cells loaded with KRAS G12V peptide. Interferon (IFN)y response was subsequently measured. Results for TCR 130 are shown in FIG. 3 and results for TCRs 240 and 243 are shown in FIG. 7. Results for the TCRs from each batch showed high sensitivity for the KRAS G12V peptide. Details of the TCRs chosen for further analysis are shown in Table 2.

[0312] Table 2: TCRs Recognizing KRAS G12V Peptide in HLA-A*02 Context

[0313] FR = framework region; CDR = complementarity determining region; Va = TCR a chain variable domain; VP = TCR P chain variable domain Once TCRs were chosen for further analysis, it was shown that these TCRs showed high transduction efficiency in host cells when co-transduced with CD8a / |3 co-receptor polypeptide and with CD8a / p co-receptor polypeptide and interleukin 7 receptor (IL7R) fusion protein (e.g., CD34-IL7R) (FIG. 9).

[0314] TCRs 130, 240, and 243 were next tested against multiple tumor models with rechallenge. T cells transduced with TCR 130 and CD8a / p co-receptor polypeptide or TCR 130, CD8a / p co-receptor polypeptide, and IL7R fusion protein were introduced to SW527 or CFPAC1 cells expressing HLA-A*02 and KRAS G12V polypeptide (FIG. 10). T cells transduced with TCR 240 / 243 and CD8a / p co-receptor polypeptide were introduced to SK- CO-1, SW480, CFPAC1, DANG, and SW527 cells expressing HLA-A*02 and KRAS G12V polypeptide (FIGs. 11-12). Each of TCRs 130. 240, and 243 showed the ability to control tumor cells even after multiple re-challenges.

[0315] Example 2: Safety Assessment

[0316] TCR 130 was also tested for off-target cytotoxicity. T cells transduced with TCR 130 were co-incubated with cholangiocellular carcinoma cell line (HuCCTl) cells expressing HLA-A*l l :01 and both of TP53 R175H and KRAS G12D peptides, as well as undifferentiated carcinoma ovarian cells, Tyknu cells, expressing HLA-A*02:01 / 26:03 and both of TP53 R175H and KRAS wild type peptides. Tumor cell confluence was measured (FIG. 5). T cells transduced with TCR 130 were shown to not kill these cell lines which did not express both KRAS G12V peptide and HLA-A*02.

[0317] TCRs 240 and 243 were tested for recognition of the wild ty pe KRAS peptide and other potential off-target interactions. First, transduced TCR 240 / 243 T cells were cocultured with T2 cells loaded with KRAS wildtype peptide. Interferon (IFN)y response was subsequently measured (FIG. 13A). The results indicated that TCRs 240 and 243 showed no recognition, or very limited recognition for wild type KRAS peptide.

[0318] Next, an X-Scan assay of TCRs 240 and 243 was conducted. In short, a KRAS peptide was individually mutated at each position to each of the 19 different amino acids and each mutated KRAS peptide thus produced was loaded into T2 cells. T cells transduced with TCRs 240 and 243 w ere introduced to the T2 cells, and IFNy response w as subsequently measured (FIG. 13B). The results showed that TCRs 240 and 243 had limited potential off- targets and confirmed the selectivity of these TCRs for KRAS G12V peptide.

[0319] Example 3: Nonvirally Modified T Cells Isolated donor T cells were modified by nonviral knock-in of constructs encoding exogenous HLA-A2 / KRAS G12V TCR130. TCR240, or TCR243 along with CD8a / p polypeptides and an ILR signaling peptide, each separated with 2A self-cleaving peptides. A schematic illustrating the knock in technology' is provided at FIG. 14. These cells were further modified to knockout endogenous TRAC and TRBC expression. For non-viral knock in and concurrent knockout of TRAC and TRBC, a nuclease system was used comprising electroporation of nuclease-gRNA RNPs to generate double-stranded breaks in the TRAC and TRBC loci. A nanoplasmid was used to provide a template for insertion of an expression cassette into the TRAC locus via homology directed repair (HDR). A preformed ribonucleoprotein (RNP) complex and nanoplasmid DNA were electroporated 48 hours after activation of T cells.

[0320] Knock-in efficiency and tetramer binding for each TCR was then analyzed (FIG. 15 and FIG. 16). Additional constructs were generated that encoded the three TCRs, CD8a / p polypeptides, and an ILR signaling polypeptide. T cells from three different donors modified with these additional knockin constructs were shown to be activated in the presence of a synthetic KRAS G12Vs-i4 peptide (FIG. 17).

[0321] T cells nonvirally modified to express either TCR130, TCR240, or TCR243 along with CD8a / p polypeptides and an ILR signaling polypeptide were then assessed for their sensitivity to HLA-A2+ / KRAS G12V+ cancer cell lines. As shown in FIGs. 18A and 18B, these TCR-T cells showed activation as measured by IFNy expression and persistence when co-mcubated with SHP77, SKCO1, SNU213, SW480, SW527, and SW620 cancer cells. These TCR-T cells also showed cytotoxic activity' against CFPAC1, SW527, SKCO1, and DAN-G cancer cell lines (FIG. 18C).

[0322] Example 4: Evaluation of TCR-engineered T cells specific for KRAS G12V mutant peptide presented by HLA-A*02:01

[0323] A non-viral knock in system was used to screen TCRs via delivery' of a transgene cassette comprising: 1) a transgenic TCR, 2) a chimeric cytokine receptor, and 3) the CD8aP coreceptor, where the transgene cassette w as knocked into the TRAC locus of human primary T cells (FIG. 19).

[0324] Specific TCR-T cell activation by KRAS G12V exogenous peptides loaded into HLA-A*02:01 was demonstrated. TCR-T cells engineered with the non-viral knock in system showed specific recognition of exogenous GI2V peptides presented by HLA-A*02:01 (FIG. 20) Processing and presentation of G12V peptide by endogenous HLA-A*02:01 was confirmed by TCR-T recognition of cell lines overexpressing KRAS G12V peptide (FIG. 21). Engineered T cells were demonstrated to potently induce functional responses to endogenous HLA-A*02:01 cancer cell lines both 1) engineered to express tandem minigenes (TMGs) encoding the KRAS G12V peptide and 2) transiently transfected with rnRNA to express full length KRAS G12V proteins. This data shows the processing and presentation of KRAS G12V peptides by endogenous HLA-A*02:01 and demonstrated its immunogenicity.

[0325] Cell lines with endogenous HLA-A*02:01 and mutant KRAS G12V were demonstrated to be sensitive to killing by the engineered TCR-T cells, but KRAS G12V negative cells were not (FIG. 22).

[0326] The recognition of HLA-A*02:01 cell lines endogenously harboring KRAS G12V mutations and the specificity of the engineered T cells was assessed by X-Scan analysis. A lack of bona fide off-targets for the engineered TCRs indicated specific on-target function of the engineered TCR-T cells (FIG. 23).

[0327] These results showed the processing and presentation of the KRAS G12V mutant peptide in the context of HLA-A*02:01 and demonstrated the feasibility of targeting this public tumor-specific neoantigen widely expressed in indications with high unmet medical need.

[0328] Other Embodiments

[0329] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0330] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0331] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A binding polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the binding polypeptide or an antigen binding portion thereof comprises one or more complementarity determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 and a heterologous constant region.

2. A T cell receptor polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the binding polypeptide or an antigen binding portion thereof comprises one or more complementarity determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 and a heterologous constant region.

3. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising TCR 130 a and / or p chains, wherein the alpha chain comprises a CDR1 comprising TSESDYY; a CDR2 comprising QEAYKQQN, and a CDR3 comprising AYNDYKLS; and the beta chain comprises a CDR1 comprising SGHVS, a CDR2 comprising FQNEAQ, and a CDR3 comprising ASSLVALAIDGELF.

4. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising TCR 240 a and / or P chains, wherein the alpha chain comprises a CDR1 comprising TRDTTYY; a CDR2 comprising RNSFDEQN, and a CDR3 comprising ALSNDYKLS; and the beta chain comprises a CDR1 comprising DFQATT, a CDR2 comprising SNEGSKA, and a CDR3 comprising SADVLAGGTDTQY.

5. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising TCR 243 a and / or P chains, wherein the alpha chain comprises a CDR1 comprising DSSSTY; a CDR2 comprising IFSNMDM, and a CDR3 comprising AETLRDTDKL1; and the beta chain comprises a CDR1 comprising DFQATT, a CDR2 comprising SNEGSKA, and a CDR3 comprising SAPRWGEQF.

6. The binding polypeptide or the T cell receptor polypeptide of claim 3, comprising a V alpha region comprising a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:a V beta region comprising a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:L.

7. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:

8. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising a beta chain comprising or consisting of a sequence having at least about 85%. at least about 90%, at least about 95%, or about 100% amino acid sequence identity’ to the following:

9. The binding polypeptide or the T cell receptor polypeptide of claim 4, comprising a V alpha region comprising a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:a V beta region comprising a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

10. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:

11. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising a beta chain comprising or consisting of a sequence having at least about 85%. at least about 90%, at least about 95%, or about 100% amino acid sequence identity’ to the following:

12. The binding polypeptide or the T cell receptor polypeptide of claim 4, comprising a V alpha region comprising a sequence having at least about 85%, at least about 90%. at least about 95%, or about 100% amino acid sequence identity to the following:a V beta region comprising a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

13. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2. comprising an alpha chain comprising or consisting of a sequence having at least about 85%, at least about 90%, at least about 95%, or about 100% amino acid sequence identity to the following:

14. The binding polypeptide of claim 1 or the T cell receptor polypeptide of claim 2, comprising a beta chain comprising or consisting of a sequence having at least about 85%, at least about 90%, at least about 95%. or about 100% amino acid sequence identity to the following:

15. The binding polypeptide or the T cell receptor of any one of claims 1-14, wherein the binding polypeptide or the antibody, or a binding portion thereof, comprises an affinity tag.

16. The binding polypeptide or the T cell receptor of any one of claims 1-15, wherein the binding polypeptide or the antibody, or a binding portion thereof, comprises a detectable amino acid sequence.

17. A polynucleotide encoding any one of the binding polypeptide or the T cell receptors of any one of claims 1-16.

18. The polynucleotide of claim 17, wherein the polynucleotide comprises a nucleic acid sequence listed in Table 1 or Table 2.

19. A vector comprising the polynucleotide of claim 17 or 18.

20. The vector of claim 19, wherein the vector is a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector.

21. A cell comprising the polynucleotide of claim 17 or 18, or the vector of claim 19 or 20.

22. A recombinant cell expressing a heterologous polynucleotide encoding a binding polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to delectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the binding polypeptide or an antigen binding portion thereof comprises one or more complementarity' determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 engineered to comprise a heterologous constant region.

23. A recombinant cell expressing a heterologous polynucleotide encoding T cell receptor polypeptide or an antigen binding portion thereof that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to delectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the binding polypeptide oran antigen binding portion thereof comprises one or more complementarity determining regions (CDRs) of a polypeptide listed in Table 1 or Table 2 engineered to comprise.

24. The recombinant cell of claim 22 or 23, wherein the cell is a T cell.

25. A composition comprising the cell of any of claims 21-24.

26. A kit comprising the binding polypeptide, the T cell receptor, or an antigen binding portion thereof, of any one of claims 1-16.

27. A kit comprising an isolated polynucleotide of claims 17 or 18.

28. A kit comprising the composition of claim 25.

29. A kit comprising the vector of claims 19 or 20 or the cell of any of claims 21-24.

30. An engineered host cell comprising a heterologous T cell receptor that specifically binds a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the heterologous T cell receptor comprises: a T cell receptor (TCR) a chain comprising an amino acid sequence with at least about 85% sequence identity' to a TCR a chain amino acid sequence of Table 1; or a TCR P chain comprising an amino acid sequence with at least about 85% sequence identity to a TCR P chain amino acid sequence of Table 1.

31. The engineered host cell of claim 30, wherein the TCR a chain comprises an amino acid sequence with at least about 90% sequence identity to a TCR a chain amino acid sequence of Table 1, or the TCR p chain comprises an amino acid sequence with at least about 90% sequence identity to a TCR P chain amino acid sequence of Table 1.

32. The engineered host cell of claim 30, wherein the TCR a chain comprises an amino acid sequence with at least about 95% sequence identity to a TCR a chain amino acid sequence of Table 1. or the TCR P chain comprises an amino acid sequence with at least about 95% sequence identity to a TCR P chain amino acid sequence of Table 1.

33. The engineered host cell of claim 30, wherein the TCR a chain amino acid sequence is a TCR a chain amino acid sequence of Table 1, or the TCR P chain amino acid sequence is a TCR P chain amino acid sequence of Table 1.

34. An engineered host cell comprising a heterologous T cell receptor that specifically binds a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the heterologous T cell receptor comprises: a TCR a chain comprising complementarity determining region (CDR) 1, CDR2. and CDR3; and a TCR P chain comprising CDR1, CDR2, and CDR3, wherein each CDR comprises an amino acid sequence having at least 85% identity to an amino acid sequence listed in Table 2.

35. The engineered host cell of claim 34, wherein each CDR comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence listed in Table 2.

36. The engineered host cell of claim 34, wherein each CDR comprises an amino acid sequence having at least about 95% sequence identity to an amino acid sequence listed in Table 2.

37. The engineered host cell of claim 34, wherein each CDR comprises or consists of an amino acid sequence listed in Table 2.

38. An engineered host cell comprising a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex, wherein the heterologous T cell receptor comprises: a T cell receptor (TCR) a chain variable (Va) domain comprising an amino acid sequence with at least about 85% sequence identity to40. The engineered host cell of claim 38, wherein the T cell receptor (TCR) a chain variable (Va) domain comprises an amino acid sequence with at least about 95% sequence identity- to41. The engineered host cell of claim 38, wherein the T cell receptor (TCR) a chain variable (Va) domain amino acid sequence is:

42. The engineered host cell of any of claims 30-41, further comprising a CD8 coreceptor.

43. The engineered host cell of any of claims 30-42. further comprising a FAS extracellular domain fused to 41 BB intracellular signaling domain and / or an IL7 receptor alpha polypeptide.

44. An engineered host cell comprising a heterologous T cell receptor that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind or binds at reduced levels to a wild-type KRAS polypeptide: HLA complex, a CD8 coreceptor, and a FAS extracellular domain fused to 41BB intracellular signaling domain and / or an IL7 receptor alpha polypeptide.

45. The engineered host cell of any of claims claim 30-44, wherein the mutant form ofKRAS peptide comprises an amino acid sequence of KLVVVGAVGV.

46. The engineered host cell of any of claims 30-45, wherein the cell is an immune cell.

47. The engineered host cell of claim 46, wherein the immune cell is a T cell.

48. The engineered host cell of claim 47, wherein the T cell comprises a CD4+T cell, aCD8+T cell, a CD4" CD8" double negative T cell, a yd T cell, or any combination thereof.

49. The engineered host cell of any of claims 30-48. further comprising a genomic mutation which causes or contributes to decreased expression of endogenous T cell receptor a constant (TRAC), T cell receptor 0 constant 1 (TRBC1), or a T cell receptor 0 constant 2 (TRBC2) of the host cell.

50. An engineered host cell comprising: a polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex; a CD8 co-receptor; and a FAS extracellular domain fused to 41BB intracellular signaling domain and / or an IL7 receptor alpha polypeptide, wherein the polynucleotide encoding the heterologous T cell receptor is inserted at a TRAC, TRBC1, or TRBC2 locus of the host cell, wherein the mutant form of KRAS peptide comprises a G12V mutation, and wherein the peptide:HLA complex comprises an HLA protein encoded by an HLA-A*02 allele.

51. An engineered host cell comprising: a heterologous TCR that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex; and a CD8 co-receptor, wherein the heterologous T cell receptor comprises a TCR a chain and a TCR 0 chain of Table 1.

52. A polynucleotide encoding a heterologous T cell receptor that specifically binds to a KRAS G12V peptide in complex with an HLA-A*02 allele, but fails to detectably bind orbinds at reduced levels to a wild-type KRAS polypeptide: HLA complex, wherein the heterologous T cell receptor comprises: a T cell receptor (TCR) a chain comprising an amino acid sequence with at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to a TCR a chain amino acid sequence of Table 1; or a TCR (3 chain comprising an amino acid sequence with at least about 85%. at least about 90%, at least about 95%, or about 100% sequence identity to a TCR [3 chain amino acid sequence of Table 1.

53. A polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex, wherein the heterologous T cell receptor comprises: a TCR a chain framework (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, CDR3, or FR4 region or a TCR 0 chain FR1, CDR1. FR2, CDR2, FR3, CDR3, or FR4 region comprising an amino acid sequence with at least about 85%. at least about 90%, at least about 95%, or about 100% sequence identity to a corresponding TCR a chain FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region or a TCR 0 chain FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region of Table 2.

54. A polynucleotide encoding a heterologous T cell receptor that specifically binds a mutant form of KRAS peptide present in a peptide:HLA complex, wherein the heterologous T cell receptor comprises: a T cell receptor (TCR) a chain variable (Va) domain comprising an amino acid sequence with at least about 85%, at least about 90%. at least about 95. or about 100% sequence identity' toP; ora TCR P chain variable (VP) domain comprising an amino acid sequence with at least about 85%. at least about 90%, at least about 95%, or about 100% sequence identity to55. The polynucleotide of any of claims 25-29, wherein the mutant form of KRAS peptide comprises an amino acid sequence of KLVVVGAVGV.

56. The polynucleotide of any of claims 52-55, wherein the polynucleotide encoding a heterologous T cell receptor is flanked by a homology7arm sequence complementary to a polynucleotide sequence encoding TRAC, TRBC1, or TRBC2, or a homology arm sequence complementary7to a polynucleotide sequence having at least 85% polynucleotide sequence identity to a genomic sequence flanking or proximal to a genomic sequence encoding TRAC, TRBC1, or TRBC2.

57. The polynucleotide of any of claims 52-56, further comprising a promoter sequence operably linked to the polynucleotide sequence encoding the heterologous T cell receptor.

58. The polynucleotide sequence of claim 57, wherein the promoter sequence is an elongation factor-1 alpha (EF-la) promoter sequence.

59. A vector comprising the polynucleotide of any of claims 52-58.

60. The vector of claim 59, wherein the vector is a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector.

61. A cell comprising the polynucleotide of any of claims 52-58, or the vector of claim 59 or 60.

62. A pharmaceutical composition comprising the engineered host cell of any one of claims 30-51 and a pharmaceutically acceptable excipient.

63. A method of treating a disease or disorder associated with a KRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of the host cell of any one of claims 30-51 or the pharmaceutical composition of claim 62.

64. A method of treating a neoplasia, the method comprising administering the engineered host cell of any one of claims 30-51 to a subject in need thereof.

65. The method of claim 63 or 64, wherein the subject is positive for an HL A- A* 02 allele.

66. The method of claim 65, wherein the subject is positive for an HLA-A*02:01 allele.

67. The method of any one of claims 63.

65. or 66, wherein the KRAS G12 mutation is a KRAS G12V mutation.

68. The method of claim 63, wherein the disease or disorder is a neoplasia.

69. The method of claim 64 or 68. wherein the neoplasia is a solid cancer70. The method of claim 64 or 68, wherein the neoplasia is a hematological malignancy.

71. The method of claim 64 or 68, wherein the neoplasia is selected from the group consisting of: a colorectal cancer, esophageal cancer, head and neck cancer, larynx cancer, lung cancer, pancreatic cancer, breast cancer, endometrial cancer, and ovarian cancer.

72. The method of claim 71 wherein the lung cancer is non-small-cell lung cancer, small cell lung cancer, or squamous cell lung cancer.

73. The method of claim 71, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

74. The method of claim 71, wherein the breast cancer is triple-negative breast cancer.

75. The method of claim 71, wherein the ovarian cancer is high-grade serous ovarian cancer.

76. The method of any one of claims 63-75, wherein the cancer is characterized as comprising a KRAS G12V mutation prior to treatment of the subject.

77. The method of any one of claims 63-76, wherein the subject is selected for treatment by characterizing a biological sample of the subject.

78. The method of any one of claims 63-77, wherein the subject has been genotyped for an HLA-A allele prior to the administering.

79. A kit comprising the host cell of any one of claims 30-51 or the pharmaceutical composition of claim 62 and instructions for use in a method of treating a neoplasia.

80. A gene cassette comprising the polynucleotide sequence of any one of claims 52-58, and a polynucleotide sequence encoding a CD8 coreceptor polypeptide and / or a polynucleotide sequence encoding a fusion protein comprising an intracellular portion of an Interleukin 7 Receptor A (IL7RA) polypeptide.

81. The gene cassette of claim 80, further comprising one or more polynucleotides encoding a self-cleaving peptide flanking one or more of the polynucleotides encoding the TCR a chain, the TCR P chain, the CD8 coreceptor polypeptide, and the fusion protein comprising an intracellular portion of the IL7RA polypeptide.

82. A cell comprising the gene cassette of claim 80 or 81.

83. A pharmaceutical composition comprising the gene cassette of claim 80 or 81, or the cell of claim 82, and a pharmaceutically acceptable excipient.