Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63/277,154, filed November 8, 2021, entitled “ENGINEERED T CELLS EXPRESSING A RECOMBINANT T CELL RECEPTOR (TCR) AND RELATED SYSTEMS AND METHODS,” the contents of which are incorporated by reference in their entirety.

Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042025440SeqList.xml, created November 4, 2022, which is 441 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

Field

[0003] The present disclosure relates to engineered T cells expressing a recombinant T cell receptor (TCR) from a T cell receptor alpha constant (TRAC) locus and also having reduced expression of transforming growth factor beta receptor 2 (TGFBR2), such as by genetic disruption at the TGFBR2 locus. Also disclosed are cell compositions containing the engineered T cells, and related methods, kits and systems for producing the engineered T cells. Also provided are methods of making and using the engineered T cells for cell therapy, including in connection with cancer immunotherapy comprising adoptive transfer of the engineered T cells.

Background

[0004] Adoptive cell therapies that utilize recombinantly expressed T cell receptors (TCRs) to recognize tumor antigens represent an attractive therapeutic modality for treatment of cancers and other diseases. In some aspects, expression and function of recombinant TCRs can be limited in a composition of engineered cells. Improved strategies are needed to achieve high expression and function of the recombinant TCRs. These strategies can facilitate generations of cells exhibiting desired properties, function or expression for use in adoptive immunotherapy, e.g., in treating cancer, infectious diseases, and autoimmune diseases. Provided herein are methods, cells, compositions, and kits for use in the methods that meet such needs. Summary

[0005] Provided herein are genetically engineered T cells expressing a recombinant T cell receptor (TCR) and compositions, methods, uses, kits, and articles of manufacture related to genetically engineered T cells. The provided embodiments, relate to T cells genetically engineered using high efficiency CRISPR-Cas9 editing as follows: 1) TRAC knock-out (KO) to prevent endogenous TCR expression; 2) knock-in (KI) of an HPV-specific recombinant TCR at the TRAC locus; and 3) KO of TGFBR2 to prevent TGFP signaling.

[0006] In some of any of the provided embodiments, the genetically engineered T cell comprises a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, and a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, wherein the genetically engineered T cell comprises a modified T cell receptor alpha constant (TRAC) locus comprising a transgene encoding the recombinant TCR or portion thereof, and has reduced expression of the gene product of the endogenous TGFBR2 locus.

[0007] In some of any of the provided embodiments, the genetically engineered T cell comprises a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, and a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; wherein the genetically engineered T cell comprises a modified T cell receptor alpha constant (TRAC) locus comprising a transgene encoding the TCRa chain and the TCRP chain of the recombinant TCR, and has reduced expression of the gene product of the endogenous TGFBR2 locus.

[0008] In some of any embodiments, the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

[0009] In some of any embodiments, the first genetic disruption is a knockout (KO) of the TGFBR2 gene. In some embodiments, all alleles of the TGFBR2 locus are knocked out in the cell. In some embodiments, both alleles of the TGFBR2 locus are knocked out in the cell. In some embodiments, expression of a gene product from the endogenous TGFBR2 locus is eliminated or prevented. [0010] In some of any embodiments, the genetically engineered T cell does not encode a functional TGFBR2 polypeptide. In some of any embodiments, the genetically engineered T cell does not encode a TGFBR2 polypeptide. In some of any embodiments, the genetically engineered T cell does not encode a full length TGFBR2 polypeptide. In some of any embodiments, the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell. In some of any embodiments, TGFP signal transmission is reduced or eliminated in the genetically engineered T cell.

[0011] In some of any embodiments, the transgene has been integrated via homology directed repair (HDR) at the TRAC locus in a cell comprising a second genetic disruption at a second target site at an endogenous TRAC locus. In some of any embodiments, the second genetic disruption is a knockout (KO) of the TRAC gene. In some embodiments, all alleles of the TRAC locus are knocked out in the cell. In some embodiments, both alleles encoding TRAC are knocked out in the cell. In some embodiments, expression of a gene product from the endogenous TRAC locus is eliminated or prevented. In some embodiments, the recombinant TCR is knocked-in (KI) to the endogenous TRAC locus. In some embodiments, the knock-in of the recombinant TCR is at or near the site of the second genetic disruption. In some of any embodiments, the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

[0012] In some of any embodiments, the first genetic disruption has been introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the second genetic disruption has been introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein. In some of any embodiments, the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein. In some of any embodiments, the Cas9 protein is a S. pyogenes Cas9 protein.

[0013] In some of any embodiments, the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129. In some of any embodiments, the first target site comprises the sequence of SEQ ID NO:83. In some of any embodiments, the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG).

[0014] In some of any embodiments, the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265. In some of any embodiments, the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55. In some of any embodiments, the second target site comprises the sequence of SEQ ID NO:238. In some of any embodiments, the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

[0015] In some of any embodiments, the integration of the transgene via HDR is carried out with a polynucleotide comprising the structure [5’ homology arm] -[transgene] -[3’ homology arm]. In some of any embodiments, the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site. In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof. In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

[0016] In some of any embodiments, the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV). In some of any embodiments, the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l l-19) YMLDLQPET (SEQ ID NO: 267). In some of any embodiments, the MHC molecule is an HLA-A2 molecule.

[0017] In some of any embodiments, the Va region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NOG, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR-3 comprising the sequence of SEQ ID NOG. In some of any embodiments, the Va region comprise the sequence of SEQ ID NOG, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOG; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some of any embodiments, the Va region comprise the sequence of SEQ ID NOG; and the VP region comprise the sequence of SEQ ID NO:1.

[0018] In some of any embodiments, the TCRa chain includes a constant alpha (Ca) region and the TCRP chain includes a constant beta (CP) region. In some of any embodiments, the Ca region and the CP region are human constant regions.

[0019] In some of any embodiments, the Ca region includes a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region includes a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183.

[0020] In some of any embodiments, the Ca region and the CP region include one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

[0021] In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2. In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2.

[0022] In some of any embodiments, the TCRa chain includes the sequence of SEQ ID NO: 14; and the TCRP chain includes the sequence of SEQ ID NO:7.

[0023] In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18.

[0024] In some of any embodiments, the transgene contains a nucleotide sequence encoding at least one further protein. In some of any embodiments, the at least one further protein comprises a surrogate marker. In some of any embodiments, the surrogate marker is a truncated receptor. In some of any embodiments, the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand.

[0025] In some of any embodiments, the transgene contains one or more multicistronic element(s). In some of any embodiments, the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain. In some of any embodiments, the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

[0026] In some of any embodiments, the one or more multicistronic element is or comprises a ribosome skip sequence. In some of any embodiments, the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element. In some of any embodiments, the one or more multicistronic element comprises a P2A element. In some of any embodiments, the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234. In some of any embodiments, the P2A element comprises SEQ ID NO:233.

[0027] In some of any embodiments, the transgene contains the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24. In some of any embodiments, the transgene contains the sequence of SEQ ID NO: 24.

[0028] In some of any embodiments, the transgene contains one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell. In some of any embodiments, the heterologous regulatory or control element comprises a heterologous promoter. In some of any embodiments, the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

[0029] In some of any embodiments, the genetically engineered T cell is less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), such as immune suppression mediated by TGFp, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cell results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cell results in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cell exhibits a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR. In some of any embodiments, the genetically engineered T cell results in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cell results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

[0030] In some of any embodiments, the disease or disorder is associated with HPV. In some of any embodiments, the disease or disorder is associated with HPV 16. In some of any embodiments, the disease or disorder is a cancer or a tumor. In some of any embodiments, the disease or disorder is a solid tumor.

[0031] In some of any embodiments, the T cell is a primary T cell derived from a subject. In some of any embodiments, the subject is a human. In some of any embodiments, the T cell is a CD8+ T cell or subtypes thereof. In some of any embodiments, the T cell is a CD4+ T cell or subtypes thereof.

[0032] Also provided herein are methods of producing genetically engineered T cells, such as any of the genetically engineered T cells described herein. In some of any embodiments, the methods involve introducing, into a T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; and introducing a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus; and introducing a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof.

[0033] In some of any embodiments, the methods involve introducing, into a T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; introducing a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus; and introducing a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region.

[0034] In some of any embodiments, the methods involve introducing, into a T cell, a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof, said T cell comprising a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

[0035] In some of any embodiments, the methods involve introducing, into a T cell, a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, said T cell comprising a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

[0036] In some of any embodiments, the first genetic disruption and the second genetic disruption is carried out by introducing, into the T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus and a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

[0037] In some of any embodiments, the polynucleotide, such as a template polynucleotide, also includes one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of a TRAC locus. In some of any embodiments, the transgene is integrated via homology directed repair (HDR) at the TRAC locus.

[0038] In some of any embodiments, the described methods produce genetically engineered T cells that comprise a modified T cell receptor alpha constant (TRAC) locus comprising the transgene encoding the recombinant T cell receptor (TCR) or portion thereof; and the first genetic disruption at the first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus and thereby reduced expression of the gene product of the endogenous TGFBR2 locus.

[0039] In some of any embodiments, the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

[0040] In some of any embodiments of the provided methods, the first genetic disruption is a knockout (KO) of the TGFBR2 gene. In some embodiments, all alleles of the TGFBR2 locus are knocked out in the cell. In some embodiments, both alleles of the TGFBR2 locus are knocked out in the cell. In some embodiments, expression of a gene product from the endogenous TGFBR2 locus is eliminated or prevented.

[0041] In some of any embodiments, the genetically engineered T cell produced by the described methods does not encode a functional TGFBRII polypeptide. In some of any embodiments, the genetically engineered T cell produced by the described methods does not encode a TGFBR2 polypeptide. In some of any embodiments, the genetically engineered T cell produced by the described methods does not encode a full length TGFBR2 polypeptide. In some of any embodiments, the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell produced by the described methods. In some of any embodiments, TGFP signal transmission is reduced or eliminated in the genetically engineered T cell produced by the described methods.

[0042] In some of any embodiments, the second genetic disruption is a knockout (KO) of the TRAC gene. In some embodiments, all alleles of the TRAC locus are knocked out in the cell. In some embodiments, both alleles encoding TRAC are knocked out in the cell. In some embodiments, expression of a gene product from the endogenous TRAC locus is eliminated or prevented. In some embodiments, the recombinant TCR is knocked-in (KI) to the endogenous TRAC locus. In some embodiments, the knock-in of the recombinant TCR is at or near the site of the second genetic disruption. In some of any embodiments, the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

[0043] In some of any embodiments, the first genetic disruption has been introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the second genetic disruption has been introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein. In some of any embodiments, the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein. In some of any embodiments, the Cas9 protein is a S. pyogenes Cas9 protein.

[0044] In some of any embodiments, the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129. In some of any embodiments, the first target site comprises the sequence of SEQ ID NO:83. In some of any embodiments, the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG). [0045] In some of any embodiments, the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265. In some of any embodiments, the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55. In some of any embodiments, the second target site comprises the sequence of SEQ ID NO:238. In some of any embodiments, the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

[0046] In some of any embodiments, the polynucleotide comprises the structure [5’ homology arm] -[transgene] -[3’ homology arm]. In some of any embodiments, the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site. In some of any embodiments, the 5’ homology arm and 3’ homology arm independently are at or about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the foregoing, or are greater than at or about 300 nucleotides in length. In some of any embodiments, the 5’ homology arm and 3’ homology arm independently are at or about 400, 500 or 600 nucleotides in length.

[0047] In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof. In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

[0048] In some of any embodiments, the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV). In some of any embodiments, the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l l-19) YMLDLQPET (SEQ ID NO: 267). In some of any embodiments, the MHC molecule is an HLA-A2 molecule.

[0049] In some of any embodiments, the Va region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NOG, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR-3 comprising the sequence of SEQ ID NOG. In some of any embodiments, the Va region comprise the sequence of SEQ ID NOG, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOG; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some of any embodiments, the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

[0050] In some of any embodiments, the TCRa chain includes a constant alpha (Ca) region and the TCRP chain includes a constant beta (CP) region. In some of any embodiments, the Ca region and the CP region are human constant regions.

[0051] In some of any embodiments, the Ca region includes a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region includes a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183.

[0052] In some of any embodiments, the Ca region and the CP region include one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

[0053] In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2. In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2.

[0054] In some of any embodiments, the TCRa chain includes the sequence of SEQ ID NO: 14; and the TCRP chain includes the sequence of SEQ ID NO:7.

[0055] In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18.

[0056] In some of any embodiments, the transgene contains a nucleotide sequence encoding at least one further protein. In some of any embodiments, the at least one further protein comprises a surrogate marker. In some of any embodiments, the surrogate marker is a truncated receptor. In some of any embodiments, the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand.

[0057] In some of any embodiments, the transgene contains one or more multicistronic element(s). In some of any embodiments, the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain. In some of any embodiments, the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

[0058] In some of any embodiments, the one or more multicistronic element is or comprises a ribosome skip sequence. In some of any embodiments, the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element. In some of any embodiments, the one or more multicistronic element comprises a P2A element. In some of any embodiments, the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234. In some of any embodiments, the P2A element comprises SEQ ID NO:233.

[0059] In some of any embodiments, the transgene contains the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24. In some of any embodiments, the transgene contains the sequence of SEQ ID NO:24.

[0060] In some of any embodiments, the transgene contains one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell. In some of any embodiments, the heterologous regulatory or control element comprises a heterologous promoter. In some of any embodiments, the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

[0061] In some of any embodiments, the polynucleotide, e.g., a template polynucleotide, is comprised in a viral vector. In some of any embodiments, the viral vector is an AAV vector. In some of any embodiments, the AAV vector is an AAV6 vector. In some of any embodiments, the viral vector is a retroviral vector. In some of any embodiments the viral vector is a lentiviral vector.

[0062] In some of any embodiments, the polynucleotide is a linear polynucleotide. In some of any embodiments, the polynucleotide is a double- stranded polynucleotide or a single-stranded polynucleotide.

[0063] In some of any embodiments, the polynucleotide is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length.

[0064] Also provided herein are systems for engineering or producing genetically engineered T cells, such as any of the genetically engineered T cells described herein. In some of any embodiments, the systems include a first agent for inducing a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus of a T cell; a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus of the T cell; and a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof; and one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of the TRAC locus.

[0065] In some of any embodiments, the systems include a first agent for inducing a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus of a T cell; a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus of the T cell; and a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region; and one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of the TRAC locus.

[0066] In some of any embodiments, the transgene is a sequence that is exogenous or heterologous to the T cell. In some of any embodiments, the first agent, the second agent and the polynucleotide are for introduction into the T cell, and the transgene is integrated via homology directed repair (HDR) at the TRAC locus.

[0067] In some of any embodiments, the introduction of the first agent, the second agent and the polynucleotide into the T cell produces a genetically engineered T cell that includes a modified T cell receptor alpha constant (TRAC) locus comprising the transgene encoding the recombinant T cell receptor (TCR) or portion thereof; and has the first genetic disruption at the first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and thereby reduced expression of the gene product of the endogenous TGFBR2 locus. [0068] In some of any embodiments, the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus. In some of any embodiments, the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

[0069] In some of any embodiments, the genetically engineered T cell does not encode a functional TGFBR2 polypeptide. In some of any embodiments, the genetically engineered T cell does not encode a TGFBR2 polypeptide. In some of any embodiments, the genetically engineered T cell does not encode a full length TGFBR2 polypeptide. In some of any embodiments, the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell. In some of any embodiments, TGFP signal transmission is reduced or eliminated in the genetically engineered T cell.

[0070] In some of any embodiments, the transgene has been integrated via homology directed repair (HDR) at the TRAC locus in a cell comprising a second genetic disruption at a second target site at an endogenous TRAC locus. In some of any embodiments, the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

[0071] In some of any embodiments, the first genetic disruption has been introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the second genetic disruption has been introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some of any embodiments, the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein. In some of any embodiments, the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein. In some of any embodiments, the Cas9 protein is a S. pyogenes Cas9 protein.

[0072] In some of any embodiments, the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129. In some of any embodiments, the first target site comprises the sequence of SEQ ID NO:83. In some of any embodiments, the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG).

[0073] In some of any embodiments, the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265. In some of any embodiments, the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55. In some of any embodiments, the second target site comprises the sequence of SEQ ID NO:238. In some of any embodiments, the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

[0074] In some of any embodiments, the polynucleotide comprises the structure [5’ homology arm] -[transgene] -[3’ homology arm]. In some of any embodiments, the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site. In some of any embodiments, the 5’ homology arm and 3’ homology arm independently are at or about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the foregoing, or are greater than at or about 300 nucleotides in length. In some of any embodiments, the 5’ homology arm and 3’ homology arm independently are at or about 400, 500 or 600 nucleotides in length.

[0075] In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof. In some of any embodiments, the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

[0076] In some of any embodiments, the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV). In some of any embodiments, the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l l-19) YMLDLQPET (SEQ ID NO: 267). In some of any embodiments, the MHC molecule is an HLA-A2 molecule.

[0077] In some of any embodiments, the Va region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region includes a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NOG, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR-3 comprising the sequence of SEQ ID NOG. In some of any embodiments, the Va region comprise the sequence of SEQ ID NOG, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOG; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some of any embodiments, the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

[0078] In some of any embodiments, the TCRa chain includes a constant alpha (Ca) region and the TCRP chain includes a constant beta (CP) region. In some of any embodiments, the Ca region and the CP region are human constant regions.

[0079] In some of any embodiments, the Ca region includes a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region includes a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183.

[0080] In some of any embodiments, the Ca region and the CP region include one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

[0081] In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2. In some of any embodiments, the Ca region includes the sequence of SEQ ID NO:9; and the CP region includes the sequence of SEQ ID NO:2.

[0082] In some of any embodiments, the TCRa chain includes the sequence of SEQ ID NO: 14; and the TCRP chain includes the sequence of SEQ ID NO:7.

[0083] In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some of any embodiments, the transgene contains a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18.

[0084] In some of any embodiments, the transgene contains a nucleotide sequence encoding at least one further protein. In some of any embodiments, the at least one further protein comprises a surrogate marker. In some of any embodiments, the surrogate marker is a truncated receptor. In some of any embodiments, the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand.

[0085] In some of any embodiments, the transgene contains one or more multicistronic element(s). In some of any embodiments, the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain. In some of any embodiments, the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

[0086] In some of any embodiments, the one or more multicistronic element is or comprises a ribosome skip sequence. In some of any embodiments, the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element. In some of any embodiments, the one or more multicistronic element comprises a P2A element. In some of any embodiments, the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234. In some of any embodiments, the P2A element comprises SEQ ID NO:233.

[0087] In some of any embodiments, the transgene contains the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24. In some of any embodiments, the transgene contains the sequence of SEQ ID NO:24.

[0088] In some of any embodiments, the transgene contains one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell. In some of any embodiments, the heterologous regulatory or control element comprises a heterologous promoter. In some of any embodiments, the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

[0089] In some of any embodiments, the polynucleotide, e.g., a template polynucleotide, is comprised in a viral vector. In some of any embodiments, the viral vector is an AAV vector. In some of any embodiments, the AAV vector is an AAV6 vector. In some of any embodiments, the viral vector is a retroviral vector. In some of any embodiments the viral vector is a lentiviral vector.

[0090] In some of any embodiments, the polynucleotide is a linear polynucleotide. In some of any embodiments, the polynucleotide is a double- stranded polynucleotide or a single-stranded polynucleotide.

[0091] In some of any embodiments, the polynucleotide is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length.

[0092] In some of any embodiments, the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein. In some of any embodiments, the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

[0093] In some of any embodiments, the concentration of the first RNP and/or the second RNP are between at or about 1 pM and at or about 5 pM, between at or about 1.5 pM and at or about 2.5 pM, between at or about 1.7 pM and at or about 2.5 pM, or between at or about 2 pM and at or about 2.5 pM. In some of any embodiments, the concentration of the first RNP and/or the second RNP are at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM.

[0094] Also provided herein are methods of producing a genetically engineered T cell that involves introducing any of the described first agent, the described second agent and/or the described polynucleotide into a T cell. Also provided herein are methods of producing a genetically engineered T cell that involves introducing the first agent, the second agent and the polynucleotide of any of the systems provided herein, into a T cell.

[0095] In some of any embodiments, the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein. In some of any embodiments, the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

[0096] In some of any embodiments, the first RNP and/or the second RNP are introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing. In some of any embodiments, the first RNP and/or the second RNP are introduced via electroporation. In some of any embodiments, the concentration of the first RNP and/or the second RNP are between at or about 1 pM and at or about 5 pM, between at or about 1.5 pM and at or about 2.5 pM, between at or about 1.7 pM and at or about 2.5 pM, or between at or about 2 pM and at or about 2.5 pM. In some of any embodiments, the concentration of the first RNP and/or the second RNP are at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM.

[0097] In some of any embodiments, the first agent and the second agent are introduced simultaneously. In some of any embodiments, the first agent and the second agent are introduced sequentially, in any order.

[0098] In some of any embodiments, the polynucleotide is introduced after the introduction of the first agent and/or the second agent. In some of any embodiments, the polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of the agent.

[0099] In some of any embodiments, prior to the introducing of the first agent and/or the second agent, the provided methods involve incubating the T cells, in vitro with one or more stimulatory agent(s) under conditions to stimulate or activate the T cells. In some of any embodiments, the one or more stimulatory agent(s) comprises anti-CD3 and/or anti-CD28 antibodies. In some of any embodiments, he one or more stimulatory agent(s) comprises anti- CD3/anti-CD28 Fab conjugated oligomeric reagent. In some of any embodiments, the provided methods also involve incubating the cells prior to, during or subsequent to the introducing of the first agent and/or the second agent and/or the introducing of the polynucleotide with one or more recombinant cytokines. In some of any embodiments, the incubation is carried out subsequent to the introducing of the first agent and/or the second agent and the introducing of the polynucleotide for up to or approximately 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. In some of any embodiments, the incubation is carried out subsequent to the introducing of the first agent and/or the second agent and the introducing of the polynucleotide for up to or approximately up to or about 7 days.

[0100] In some of any embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus. In some of any embodiments, at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR. In some of any embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods do not express a gene product of an endogenous TRAC locus.

[0101] In some of any embodiments, at least at or about 80% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods do not express a gene product of an endogenous TRAC locus.

[0102] In some of any embodiments, the T cell is a primary T cell derived from a subject, optionally wherein the subject is a human. In some of any embodiments, the T cell is a CD8+ T cell or subtypes thereof. In some of any embodiments, the T cell is a CD4+ T cell or subtypes thereof.

[0103] In some of any embodiments, the genetically engineered T cells produced any of the described methods, are less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), optionally immune suppression mediated by TGFp, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, exhibit a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

[0104] In some of any embodiments, the disease or disorder is associated with HPV, optionally HPV 16. In some of any embodiments, the disease or disorder is a cancer or a tumor, optionally a solid tumor.

[0105] Also provided herein are genetically engineered T cells generated using any of the provided methods.

[0106] Also provided herein are compositions that include any of the genetically engineered T cells provided herein.

[0107] Also provided herein are compositions that include a plurality of any of the genetically engineered T cells provided herein.

[0108] In some of any embodiments, the composition also includes a pharmaceutically acceptable excipient.

[0109] In some of any embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells in any of the provided compositions comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus. In some of any embodiments, at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells in any of the provided compositions express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR. In some of any embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells in any of the provided compositions do not express a gene product of an endogenous TRAC locus. In some of any embodiments, at least at or about 80% of the engineered T cells in any of the provided compositions comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells in any of the provided compositions express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells in any of the provided compositions do not express a gene product of an endogenous TRAC locus.

[0110] In some of any embodiments, the composition comprises CD4+ T cells and/or CD8+ T cells. In some of any embodiments, the composition comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition; and/or the percentage of CD8+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition. In some of any embodiments, the composition comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1, optionally at or about 1:1.

[0111] Also provided are methods of treating a disease or disorder, such as therapeutic methods, and uses, such as therapeutic uses, of any the provided genetically engineered T cells, pluralities thereof or compositions comprising any of the provided genetically engineered T cells. [0112] Also provided are methods of treating a disease or disorder that involves administering of any of the provided genetically engineered T cells, pluralities of any the provided genetically engineered T cells, or compositions comprising any of the provided genetically engineered T cells, to a subject having the disease or disorder.

[0113] Also provided are uses of any of the provided genetically engineered T cells, pluralities of any the provided genetically engineered T cells, or compositions comprising any of the provided genetically engineered T cells for treating a disease or disorder.

[0114] Also provided are uses of any of the provided genetically engineered T cells, pluralities of any the provided genetically engineered T cells, or compositions comprising any of the provided genetically engineered T cells in the manufacture of a medicament for treating a disease or disorder.

[0115] Provided herein are any of the provided genetically engineered T cells, pluralities of any the provided genetically engineered T cells, or compositions comprising any of the provided genetically engineered T cells, for use in treating a disease or disorder.

[0116] In some of any embodiments, the disease or disorder is associated with HPV, optionally HPV 16.

[0117] In some of any embodiments, the disease or disorder is a cancer or a tumor, optionally a solid tumor. In some of any embodiments, the tumor is associated with a cervical cancer, a uterine cancer, an anal cancer, a colorectal cancer, a vaginal cancer, a vulvar cancer, a penile cancer, a oropharyngeal cancers, a tonsil cancer, a pharyngeal cancers, a laryngeal cancer, an oral cancer, a skin cancer, a esophageal cancer, a head and neck cancer or a small cell lung cancer. In some of any embodiments, the tumor is associated with a head and neck cancer, optionally a head and neck squamous cell carcinoma (HNSCC). In some of any embodiments, the tumor is associated with a cervical cancer, optionally a cervical carcinoma.

[0118] In some of any embodiments, a dose of the genetically engineered T cells is administered to the subject.

[0119] In some of any embodiments, the dose of genetically engineered T cells comprises between at or about 3 x 10 ⁷ recombinant TCR-expressing T cells and at or about 3 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive. In some of any embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR- expressing T cells and at or about 1 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive. In some of any embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR-expressing T cells and at or about 1 x 10 ⁹ recombinant TCR- expressing T cells, inclusive. In some of any embodiments, the dose of genetically engineered T cells contains at or about 1 x 10 ⁸ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR-expressing T cells; at or about 1 x 10 ⁹ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR-expressing T cells; or at or about 1 x IO ¹⁰ recombinant TCR-expressing T cells

[0120] In some of any embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10 ⁸ recombinant TCR-expressing T cells. In some of any embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10 ⁸ recombinant TCR-expressing T cells. In some of any embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10 ⁹ recombinant TCR-expressing T cells. In some of any embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10 ⁹ recombinant TCR-expressing T cells. In some of any embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10 ¹⁰ recombinant TCR-expressing T cells.

[0121] In some of any embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and/or CD 8+ T cells.

[0122] In some of any embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and/or the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose. In some of any embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1, optionally at or about 1:1.

[0123] In some of any embodiments, interleukin-2 (IL-2) or a variant thereof is further administered to the subject.

[0124] In some of any embodiments, the dose of genetically engineered T cells, are less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), optionally immune suppression mediated by TGFp, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, exhibit a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder. In some of any embodiments, the genetically engineered T cells produced any of the described methods, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

Brief Description of the Drawings

[0125] FIG. 1A depicts cell surface expression of CD4 and CD8 (top panel) and exemplary recombinant TCR-specific Vbeta staining and CD3 (bottom panel), as assessed by flow cytometry, for T cells engineered as follows: engineered to express an exemplary anti-HPV 16 TCR1 by targeted integration (knock-in) at an endogenous TCR encoding gene and engineered with a knockout of endogenous TGFBR2 (TCR1 TRAC KI TGFBR2 KO) or without a knockout of endogenous TGFBR2 (TCR1 TRAC KI TGFBR2 WT), or with a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO), or knockout of an endogenous TCR encoding gene alone (TRAC KO). FIG. IB depicts cell surface expression of MHC-peptide tetramer complexed with the antigen recognized by the recombinant TCR and Vbeta2, as assessed by flow cytometry, for CD4+ or CD8+ T cells in mock-transduced (left four panels) and transduced (left right panels) T cells subject to knockout of endogenous TCR encoding gene (TRAC), engineered to express TCR1 (TCR1 TRAC KI; top panels) or without a knock-in of TCR1 (TCR1 TRAC KO; bottom panels).

[0126] FIGS. 2A-2D depict the changes in tumor spheroid size after incubation with T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of the endogenous TGFBR2 locus. The cells were stimulated with HNSCC HPV+ SCC152 tumor spheroids at E:T ratio of 1:2, 1:10 or 1:20 for 10 days, followed by restimulation with tumor spheroids on Day 10, and monitoring of tumor spheroid size until Day 20. FIG. 2A shows tumor spheroid size plotted as time post-restimulation with tumor spheroids (Day 10) and represents cytotoxic activity from Days 10 to 20, with smaller tumor spheroid size representing greater anti-tumor activity. FIG. 2B depicts representative images showing the reduction in spheroid size (spheroids indicated by reduced fluorescence). FIG. 2C (optimal E:T; 1:2) and FIG. 2D (sub-optimal E:T; 1:20) depict tumor spheroid size plotted as time post- restimulation with tumor spheroids (Day 10), comparing the changes at an optimal E:T ratio or a sub-optimal E:T ratio.

[0127] FIGS. 3A-3C depict expression of cell differentiation markers for T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2. FIG. 3A shows cell surface expression of CD101 and CD38, as assessed by flow cytometry, for each group. FIG. 3B shows the percentage of CD101+ CD38+ T cells for each group. FIG. 3C shows geometric mean fluorescence intensity (gMFI) for expression of CD25, CD27, PD-1, and TIM-3 for each group. A quantification (top panel) of a flow cytometry histogram (bottom panel) is shown.

[0128] FIG. 4A depicts representative images showing the reduction in spheroid size (spheroids indicated by reduced fluorescence) after incubation with T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, at day 20 of the re- stimulation study, and at a sub-optimal E:T ratio of 1:20.

[0129] FIGS. 4B-4C depict the number of live cells and cell expansion for T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus after initial stimulation (FIG. 4B) and after restimulation with spheroids (FIG. 4C).

[0130] FIG. 4D depict the level of cytokines interferon-gamma (IFN-y), interleukin-2 (IL- 2), and tumor necrosis factor-alpha (TNF-a) produced in the tumor spheroid co-culture, at days 3, 10 and 20, at E:T ratios of 1:2, 1:10 or 1:20, as measured by a multiplexed cytokine assay kit.

[0131] FIGS. 5A-5B depict measurement of in vivo anti-tumor effects in CaSki or SCC152 tumors isolated from NOD.Cg-Prkdc ^scld I12rg ^{tml wjl} /SzJ (NSG) mice injected with squamous cell carcinoma cell line UPCESCC-152 (ATCC® CRL-3240™) cells, originating from a recurrent squamous cell carcinoma of the hypopharynx, that are HLA-A*02:01-positive, HPV-16- positive, and express E7 HPV oncoprotein. FIG. 5A shows TGFP levels at staging measured in CaSki or SCC152 tumor samples. FIG. 5B shows the percentage of cells that exhibit surface expression of CD 103 among CD3+ cells and tumor infiltrating lymphocytes (TIL) that express the recombinant TCR (TCR1) at day 7 and day 14 after administration of the engineered cells.

[0132] FIGS. 6A-6E depict anti-tumor activity at different doses of T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, or T cells with a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO), at a dose of 3.33 x 10 ⁵ cells (FIG. 6A) and 1 x 10 ⁶ cells (FIG. 6B). or a control without T cell addition. Mean tumor volume was measured. FIG. 6C shows mean tumor volume measured in individual mice receiving a dose of 3.33 x 10 ⁵ cells. Modified Tumor Control Index (mTCI) score was assessed for mice administered a dose of 3.33 x 10 ⁵ cells (FIG. 6D) and 1 x 10 ⁶ cells (FIG. 6E). FIG. 6F shows percentage of tumor-free survival in individual mice. Statistical significance values are reported as q (adjusted p value): *** p < 0.0001; ns = not significant.

[0133] FIGS. 7A-7B depict cell expansion of T cells subject to knockout of endogenous TCR encoding gene (TRAC), engineered to express TCR1 and engineered with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, or a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO) or a control without T cell additions as measured by circulating CD4+ (FIG. 7A) and CD 8+ (FIG. 7B) T cells in blood.

[0134] FIGS. 8A-8B depict the percentage of CD 103 -expressing cells among CD3+ TCR+ cells, as assessed by flow cytometry, of T cells subject to knockout of endogenous TCR encoding gene (TRAC), engineered to express TCR1 and engineered with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, at a dose of 3.33 x 10 ⁵ cells (FIG. 8A) or at a dose of 1 x 10 ⁶ cells (FIG. 8B).

[0135] FIGS. 9A-9D depict measurement of in vivo anti-tumor effects of T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, or a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO) injected cells in cervical cancer model generated by subcutaneous injection of CaSki human cervical cancer cell line, originating from a metastasis in the small bowel mesentery, into NOD.Cg- Prkdc ^scld n2rg ^tmlwjl /SzJ (NSG) mice. A tumor only group was used as control. FIG. 9A shows mean tumor volume. FIG. 9B shows mean tumor volume of individual mice per group. FIG. 9C shows modified Tumor Control Index (mTCI) score of mice in each group. FIG. 9D shows percentage of tumor- free survival of mice in each group.

[0136] FIGS. 10A-10C depict cell expansion of T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, or a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO) as measured by circulating CD4+ (FIG. 10A) and CD8+ (FIG. 10B) T cells in blood. A tumor only group was used as control. FIG. 10C shows the percentage of CD103-expressing cells among CD3+ TCR+ cells, as assessed by flow cytometry at day 14.

[0137] FIG. 11 is a schematic representation of exemplary anti-HPV TCR-expressing cells by targeted knock-in at the endogenous TRAC locus, with a knockout of the TGFBR2 gene (TCR1 TRAC KI TGFBR2 KO), or the same exemplary anti-HPV TCR without the knockout of TGFBR2 gene (TCR1 TRAC KI TGFBR2 WT).

[0138] FIG. 12 depicts TGFp levels in CaSki, SCC152 or SCC104 tumor samples.

[0139] FIGS. 13A-13C depict the mean tumor volume changes over time, representing antitumor activity, in a mouse tumor model of head and neck squamous cell carcinoma (HNSCC) expressing higher levels of TGFP (UM-SCC-104), administered T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus, a knockout of an endogenous TCR encoding gene and a knockout of an endogenous TGFBR2 locus (TRAC KO TGFBR2 KO), at a dose of 6 x 10 ⁶ cells (FIG. 13A), 2 x 10 ⁶ cells (FIG. 13B), or 6 x 10 ⁵ cells (FIG. 13C), or a control without T cell addition (tumor only). FIGS. 14A-14C depict the tumor volume changes over time in individual UM- SCC-104 mice administered TCR1 TRAC KI TGFBR2 KO, TCR1 TRAC KI TGFBR2 WT, TRAC KO TGFBR2 KO, at a dose of 6 x 10 ⁶ cells (FIG. 14A), 2 x 10 ⁶ cells (FIG. 14B), or 6 x 10 ⁵ cells (FIG. 14C), or a control without T cell addition (tumor only).

[0140] FIGS. 15A-15C depict the modified Tumor Control Index (mTCI) score in UM- SCC-104 mice administered TCR1 TRAC KI TGFBR2 KO, TCR1 TRAC KI TGFBR2 WT, TRAC KO TGFBR2 KO, at a dose of 6 x 10 ⁶ cells (FIG. 15A), 2 x 10 ⁶ cells (FIG. 15B), or 6 x 10 ⁵ cells (FIG. 15C), or a control without T cell addition (tumor only).

[0141] FIGS. 16A-16C depict the survival curve of UM-SCC-104 mice administered TCR1 TRAC KI TGFBR2 KO, TCR1 TRAC KI TGFBR2 WT, TRAC KO TGFBR2 KO, at a dose of 6 x 10 ⁶ cells (FIG. 16A), 2 x 10 ⁶ cells (FIG. 16B), or 6 x 10 ⁵ cells (FIG. 16C), or a control without T cell addition (tumor only).

[0142] FIG. 17 depict the percentage of CD 103 -expressing cells among CD3+ TCR+ cells, as assessed by flow cytometry, in UM-SCC-104 mice administered T cells engineered to express the exemplary anti-HPV TCR1 by targeted knock-in at the endogenous TRAC locus, with (TCR1 TRAC KI TGFBR2 KO) or without (TCR1 TRAC KI TGFBR2 WT) a knockout of endogenous TGFBR2 locus.

Detailed Description

[0143] Provided herein are genetically engineered cells, such as T cells, expressing a recombinant receptor, such as a recombinant T cell receptor (TCR), that binds or recognizes a peptide epitope associated with a cancer antigen or a tumor antigen. In particular embodiments, the recombinant TCR targets an HPV antigen, such as the tumor restricted HPV 16 E7( 11 - 19) onco-peptide, e.g. the recombinant TCR designated TCR1 in the present disclosure. In some aspects, the engineered T cell expressing the recombinant TCR comprises a genetic disruption to reduce, such as eliminate, the expression of the gene product of a transforming growth factor beta receptor 2 (TGFBR2) locus, and a modified gene that encodes a domain or region of a T cell receptor, such as a modified T cell receptor alpha constant (TRAC) locus, comprising a transgene encoding the recombinant TCR or portion thereof. Also provided are methods for engineering, preparing, and producing the engineered cells, kits and articles of manufacture for generating or producing the engineered cells. Also provided are polynucleotides that contain a transgene encoding a recombinant TCR or a portion thereof, and methods for introducing such polynucleotides into the cells, such as by transduction or by physical delivery, such as electroporation, and vectors for introducing such polynucleotides. Also provided are compositions containing the engineered cells, methods, kits for producing and administering the cells and compositions to subjects, and methods and uses, including therapeutic methods and uses, of the engineered cells and cell compositions, such as for cell therapy.

[0144] In some aspects, expression of the endogenous TGFBR2 locus (encoding the TGFBR2 protein) is knocked out, reduced or eliminated, in the engineered cell, by virtue of the genetic disruption (such as a knock-out (KO)) at the TGFBR2 locus. In some aspects, the recombinant TCR or a portion thereof, is encoded by transgene sequences that are integrated at a TRAC locus in the genome of the cell, resulting in a modified TRAC locus in the genome. In some aspects, the integration of the transgene is achieved by inducing a targeted genetic disruption, e.g., generation of a DNA break, using gene editing methods, and homology-directed repair (HDR) for targeted knock-in (KI) of the recombinant TCR-encoding transgene at a gene locus encoding the endogenous TCR (e.g., TRAC), thereby reducing or eliminating the expression of the endogenous TCR, at the same time allowing for expression of the recombinant (and typically exogenous or heterologous) TCR. Accordingly, the modified TRAC locus comprising a transgene encoding the recombinant TCR or portion thereof can facilitate a uniform or homogeneous expression of the recombinant TCR within a cell population, at the same time reducing or eliminating the expression of the endogenous TCR.

[0145] Among the provided embodiments are approaches useful in the treatment of human papillomavirus (HPV)-related diseases and conditions and/or for targeting such cell types, such as cancer cells or cells that are infected with HPV or cells that contains HPV DNA sequences. In some embodiments, the recombinant TCRs bind or recognize a peptide epitope of HPV, such as HPV 16 E7, in the context of an MHC molecule. In some aspects, the engineered cells expressing a recombinant TCR can specifically target and kill such cells expressing HPV or associated with HPV-related diseases and conditions. For instance, the provided embodiments relate to engineering T cells to express a recombinant TCR against the tumor restricted HPV 16 E7(l 1-19) onco-peptide. Among such TCRs is the recombinant TCR designated TCR1 (see, e.g., Table 8, and containing a Va region set forth in SEQ ID NO:8 and a VP region set forth in SEQ ID NO:1, such as the TCRa sequence set forth in SEQ ID NO: 14 and the TCRP sequence set forth in SEQ ID NO:7).

[0146] Adoptive cell therapy with transfer of recombinant receptor-expressing T cells targeting cell surface antigens (e.g., chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR)) has shown success in hematological malignancies. However, effectively targeting solid tumors has been limited, in part due to challenges in identifying highly expressed, tumor specific antigens and the immune suppressive tumor microenvironment mediated by cellular and secreted factors such as TGFp, known to suppress intra-tumoral immunity and substantially elevated in many human cancers, including in human papilloma virus-associated cancers (e.g., head and neck squamous cell carcinoma and cervical cancers). Improved strategies are needed.

[0147] Provided are genetically engineered T cells, compositions and related methods and uses that meet such needs. In some aspects, the provided embodiments are based on observations as exemplified in the Examples, of highly potent, TGFP armored (e.g., by reduced expression of TGFBR2 via a genetic disruption, such as a knock-out (KO), at the TGFBR2 locus), engineered T cells expressing a recombinant fully human TCRaP sequence (for example, TCR1 as referred to in the disclosure) that targets the tumor restricted HPV 16 E7(l 1-19) onco- peptide. In some aspects, the provided embodiments relate to T cells genetically engineered by genetic disruption (such as a knock-out (KO)) at the endogenous gene encoding TCRa constant domain (TRAC) to reduce or eliminate the expression of the endogenous TCR alpha chain constant region (Ca), together with a targeted insertion (such as a knock-in (KI)) of the recombinant TCR (e.g., anti-HPV 16 E7(l l-19) TCR, such as TCR1) encoding transgene at the TRAC locus, and, a genetic disruption (such as a knock-out (KO)) at the endogenous TGFBR2 locus to prevent TGFP signaling. In some embodiments, compositions containing a plurality of the engineered T cells can be generated by high-efficiency CRISPR/Cas9 combination gene editing, including from donor-derived T cells obtained from subjects, such as human subjects with HPV-associated cancers (e.g. head and neck squamous cell carcinoma and cervical cancers).

[0148] As exemplified in the disclosure, compositions comprising the engineered T cells, characterized using flow cytometry and molecular techniques, were shown to exhibit more than 95% TRAC KO, more than 80% TGFBR 2 KO and more than 75% anti-HPV recombinant TCR expression (e.g., TCR1 as referred to in the disclosure that targets the tumor restricted HPV 16 E7(l 1-19) onco -pep tide). As exemplified herein, pharmacology studies demonstrated that the engineered cells expressing the recombinant anti-HPV TCR and with TGFP armoring (e.g., by reduced expression of TGFBR2 via a genetic disruption at TGFBR2) to overcome TGFP- mediated immune suppression was highly effective, even in sub-optimal conditions, such as exemplified both in vitro using a 3D serial spheroid stimulation and in vivo using mouse tumor xenografts against at least three cancer lines, SCC-152, SCC-104, and CasKi. Also, in some cases, at sub-optimal effector to T cell (E:T) ratios, the provided engineered T cells exhibit superior expansion, cytotoxicity and an improved functional phenotype. For example, the engineered T cells exhibit reduced CD 103 staining (surrogate marker downstream of the TGFP activation pathway) both in in vitro and in vivo tumor models compared to non-armored engineered T cells (e.g., without TGFBR2 KO). As observed by the improvements in properties, including improvements in pharmacodynamics and phenotypic characteristics, the provided engineered T cells and compositions support an effective clinical applications in solid tumors, for example, even at lower doses.

[0149] T cell-based therapies, such as adoptive T cell therapies (including those involving the administration of engineered cells expressing recombinant, engineered or chimeric receptors specific for a disease or disorder of interest, such as a recombinant T cell receptor (TCR) or other recombinant, engineered or chimeric receptors) can be effective in the treatment of cancer and other diseases and disorders. In certain contexts, other approaches for generating engineered cells for adoptive cell therapy may not always be entirely satisfactory. In some aspects, efficacy or potency of the engineered cells can depend on various factors, including T cell exhaustion, immunosuppressive tumor microenvironment (TME), poor cell infiltration into the target, e.g., tumor, lack of endogenous anti-tumor immune response, and poor expression of the recombinant receptor, mispairing or competition with endogenously expressed TCRs. [0150] In some contexts, optimal activity or outcome can depend on the ability of the administered cells to express the recombinant receptor, e.g., recombinant TCR, on the surface, recognize and bind to a target, e.g., target antigen, to traffic, localize to and successfully enter appropriate sites within the subject, tumors, and environments thereof. In some contexts, optimal activity or outcome can depend on the ability of the administered cells to uniformly and/or continuously express the recombinant receptor, become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states (such as long-lived memory, less-differentiated, and effector states), to avoid or reduce immunosuppressive conditions in the local microenvironment of a disease, to provide effective and robust recall responses following clearance and re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, peripheral tolerance, terminal differentiation, and/or differentiation into a suppressive state.

[0151] In some aspects, the provided embodiments offer an advantage that allows engineered cells administered for adoptive therapy to alleviate or overcome immunosuppressive effects of TGFP in the tumor microenvironment (TME). In some cases, the TME contains or produces factors or conditions, such as TGFp, that can mediate immunosuppressive signals to suppress the activity, function, proliferation, survival and/or persistence of T cells administered for T cell therapy. In some contexts, binding of the ligand transforming growth factor beta (TGFP) to an endogenous TGFBR2, which is a receptor normally expressed on the surface of immune cells, such as T cells, initiates formation of a receptor complex to initiate cellular signaling. TGFP-mediated cellular signaling in immune cells, such as CD4+ and CD8+ T cells, can result in suppression of CD8+ T cells and induction of regulatory T cell (Treg) phenotypes in CD4+ cells. In some aspects, TGFP in the TME can affect T cell proliferation, inhibit the maturation of T helper cells and/or reduce T cell effector function. In some aspects, TGFP can repress the expression of genes involved in cytotoxicity in T cells, such as perforin, granzyme A, granzyme B, IFNy and Fas ligand. In some aspects, TGFP can induce the development of Treg cells that can result in immunosuppression. In some aspects, reduction or downregulation of TGFP mediated cellular signaling, e.g., by knock-out of expression of a receptor for TGFP such as TGFBR2, or expression of a dominant-negative form of TGFBR2, can result in overcoming suppressive effects of TGFP signaling in cells (see, e.g., Yang et al., Trends Immunol. (2010) 31(6): 220-227; Oh et al., J Immunol. (2013) 191(8): 3973-3979; Principe et al., Cancer Res. (2016) 76(9): 2525-2539). In some embodiments, reduction or elimination of expression of TGFBR2 in the engineered cell permit the engineered cells to alleviate or overcome the immnosuppressive effects, such as immunosuppressive effects of TGFP-mediated signaling, and promote the function, activity, proliferation, survival and/or persistence of T cells.

[0152] In some embodiments, the provided embodiments involve inducing a genetic disruption at the endogenous TGFBR2 locus, thereby altering, reducing or eliminating the expression of TGFBR2 from the endogenous TGFBR2 gene. In some aspects, the provided embodiments are based on observations that reduction and/or elimination of expression of TGFBR2, for example by a genetic disruption (e.g., knock-out) results in improved activity and/or function, such as anti-tumor activity, cytokine production, expansion and/or persistence, of the engineered cells expressing a recombinant receptor, such as a recombinant TCR. In some aspects, the engineered cells in which the expression of TGFBR2 is knocked out, reduced or eliminated, or a modified form of TGFBR2 polypeptide is expressed. In some embodiments, the provided polynucleotides, transgenes, and/or vectors, when delivered into immune cells, result in the expression of recombinant TCR that can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis.

[0153] In some contexts, the provided methods can be used in connection with solid tumor targets or other disease microenvironments where TGFP immunosuppressive activity may otherwise impair or reduce the function, survival or activity of a T cell therapy. Moreover, the provided cells, compositions, nucleic acids, kits and methods also offer advantages in controlling and regulating expression of the recombinant TCR on cells of the T cell therapy. The resulting genetically engineered cells or cell compositions can be used in adoptive cell therapy methods.

[0154] In some aspects, the provided embodiments are also based on observations of improved expression of an exemplary fully human recombinant TCR, such as certain provided TCRs specific to HPV 16 E7 (for example, TCR1 as referred to in the disclosure that targets the tumor restricted HPV 16 E7(l 1-19) onco-peptide), and improved activity of engineered T cells expressing the exemplary recombinant TCR at the modified TRAC locus and with a genetic disruption at TGFBR2 (e.g., reducing or eliminating expression of the TGFBR2 gene product), even at a low effector to target (E:T) ratio or a low dose administration (e.g., a sub-optimal E:T ratio or a sub-optimal dose). The activity of the engineered T cells expressing a recombinant TCR, e.g., cytolytic activity, in some cases may be limited when fewer engineered T cells are present compared to the target cells (e.g., cancer cells expressing the antigen). In some aspects, such improvements in activity, particularly at a low E:T ratio and using fully human sequences, are advantageous in improving the efficacy of the therapy, e.g., adoptive cell therapy. In certain aspects, the substantial improvement in anti-tumor activity were observed at a low E:T ratio or a low dose of administration as described herein, support an improved therapeutic effect even at a low dose of administration. Further, the provided embodiments were also observed to result in improved expansion of engineered cells in the presence of the target (e.g., HPV-expressing target cells) both in vitro and in vivo, even in a prolonged exposure. The engineered cells were also observed to exhibit lower levels of indicators of terminal differentiation and exhaustion.

[0155] Taken together, such improvements of the engineered T cells as provided herein support administration of a reduced dose of cells to achieve a therapeutic effect. This is advantageous compared to other engineered TCR products, including those directed against HPV, in which efficacy is generally reported at high doses (>10 billion cells) which, in some aspects, may not be commercially viable. For instance, the ability to generate an engineered T cell product that supports a dosage administration of at or about or less than 10 billion recombinant TCR-expressing T cells can reduce the time and cost associated with manufacturing or preparing the engineered cells for cell therapy. In some cases a lower dose also may reduce potential adverse effects of cell therapy. In some embodiments, compositions containing the provided engineered T cells can be used for treating an HPV-associated cancer at a dosage administration of between 1 x 10 ⁸ recombinant TCR-expressing T cells and 1 xlO ¹⁰ recombinant TCR-expressing T cells, for instance at or about IxlO ⁹ recombinant TCR- expressing T cells or IxlO ⁸ recombinant TCR-expressing T cells. In some embodiments, the number of TCR-expressing T cells in a population of engineered T cells is determined by flow cytometry. In some embodiments, the recombinant TCR-expressing T cells are the number of such viable cells (e.g. viable cell concentration, cells/mL; or cell viability, % viable), such as determined using an automated cell counter.

[0156] In some aspects, as described herein, the engineered cells include a genetic disruption at an endogenous gene encoding a domain, chain or region of the endogenous TCR, for example, an endogenous TCR alpha constant (TRAC) locus, thereby reducing or eliminating the expression of the endogenous TRAC gene product. In some aspects, a transgene sequence (also referred to herein as exogenous or heterologous nucleic acid sequences) encoding all or a portion of a recombinant TCR is integrated at an endogenous TRAC locus, which normally encodes a TCRa constant domain.

[0157] In some embodiments, provided herein are methods of generating or producing genetically engineered cells that contain TRAC locus includes nucleic acid sequences encoding a recombinant TCR or a portion thereof, such as a TCR alpha (TCRa) chain or a TCR beta (TCRP) chain of a recombinant TCR. In some embodiments, the methods involve inducing a targeted genetic disruption and homology-directed repair (HDR), using one or more polynucleotides (e.g., a template polynucleotide) containing the transgene encoding all or a portion of the recombinant TCR, thereby targeting integration of the transgene at the TRAC locus. Also provided are cells and cell compositions generated by the methods. In some aspects, elimination of expression of the endogenous TCRa chain can reduce mispairing between an endogenous and the engineered or recombinant chains.

[0158] The provided embodiments also offer advantages in producing engineered T cells, where all cells that express the recombinant TCR are also knocked out for, reduced and/or eliminated the expression of an endogenous TCR gene locus (such as the endogenous genes encoding the TCRa chain) via gene editing and HDR. Compared to approaches that may produce a heterogeneous mixture, where some of the cells that express the recombinant TCR may be knocked out for the endogenous TCR gene loci while other cells that express the recombinant TCR may retain the endogenous TCR gene loci, the provided embodiments can be used to generate a substantially more homogeneous and uniform population of cells, e.g., where all cells that express the recombinant TCR contain knock-out of an endogenous TCR gene locus.

[0159] In some aspects, the provided embodiments are also based on observations of improved efficiency of integration and expression and antigen binding of TCRs using the targeted knock-in approach. Targeted knock-out of an endogenous TCR gene locus (such as the endogenous genes encoding the TCRa chain) by gene editing, combined with targeted knock-in of nucleic acids encoding the recombinant TCR by homology-directed repair (HDR), can facilitate the production of engineered T cells that are improved in expression, function and uniformity of expression and/or other desired features or properties, and ultimately high efficacy.

[0160] In some aspects, compared to conventional methods of producing genetically engineered immune cells expressing a recombinant TCR, the provided methods allow for a higher, much more stable and/or much more uniform or homogeneous expression of the recombinant TCR. In some aspects, the provided embodiments offer advantages in producing engineered T cells with improved, uniform, homogeneous, consistent and/or stable expression of the recombinant TCR, while minimizing possible mispairing, mis-targeting, semi-random or random integration of the transgene and/or competition from endogenous TCRs. For example, in some cases, suboptimal expression of an engineered or recombinant TCR can occur due to competition with an endogenous TCR and/or with TCRs having mispaired chains, for signaling molecules and/or domains such as the invariant CD3 signaling molecules (e.g., availability of co-expressed co-expression of CD3 6, a, y and C, chains) that are involved in permitting expression of the complex on the cell surface. In some aspects, available CD3(^ molecules can limit the expression and function of the TCRs in the cells. In some aspects, currently available methods for delivery of transgenes, e.g., encoding recombinant TCR may show inefficient integration and/or reduced expression of the recombinant TCRs. In some aspects, the efficiency of integration and/or expression of the recombinant TCR within a population may be low and/or varied.

[0161] In some aspects, the provided embodiments also permit predictable and consistent integration at a single gene locus or a multiple gene loci of interest, provide consistent copy number (typically, 1 or 2) of the nucleic acids, have reduced, low or no possibility of insertional mutagenesis, provide consistency in recombinant TCR expression and reduction of expression of the endogenous TCR genes within a cell population, and eliminate the requirement for RCL assays. In some embodiments, the provided compositions exhibit reduced coefficient of variation of expression and/or antigen binding, compared to that of cell populations and/or compositions generated using conventional methods. In some aspects, the provided embodiments are based on observations that targeted knock-in of the recombinant TCR- encoding nucleic acids at an endogenous TCR gene locus, e.g., TRAC, which reduces or eliminates the expression of the endogenous TCR genes, resulted in a higher overall level of expression, a more uniform and consistent expression and/or antigen binding, and improved function of the engineered cells, including improved anti-tumor effects.

[0162] In some embodiments, certain available recombinant TCRs exhibit cross reactivity to a different, non-target antigen (see, e.g., Cameron et al., (2013) Science Translational Medicine, 5(197): 197ral03). In some aspects, the provided embodiments employ fully human recombinant TCR, such as certain provided TCRs specific to HPV E7, that do not show cross reactivity to cells expressing other peptide antigens or alloreactivity to other human leukocyte antigen (HLA) subtypes. The TCRs thus also exhibit improved expression and activity, with minimal risk of cross reactivity to other antigens, such as non-target antigens, that can be present in the subject, or peptide epitopes present on non-target HLA subtypes. Accordingly, the described embodiments provide numerous advantages over conventional methods of adoptive cell therapy.

[0163] Also provided are nucleic acid molecules encoding the recombinant TCRs, engineered cells containing the recombinant TCRs, compositions containing the recombinant TCRs or cells, and methods of treatment involving administering such recombinant TCRs, engineered cells or compositions, and uses of such recombinant TCRs, cells or compositions. In some aspects, engineered cells that express a provided recombinant TCR, e.g. a TCR or antigenbinding fragment thereof, exhibit cytotoxic activity against target cells expressing the peptide epitope, such as cancer cells or cells that are infected with HPV.

[0164] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0165] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. CELLS EXPRESSING A RECOMBINANT T CELL RECEPTOR (TCR) ENGINEERED BY GENE EDITING AND HOMOLOGY-DIRECTED REPAIR

[0166] Provided herein are engineered T cells that express a recombinant T cell receptor (TCR). In some aspects, the provided engineered T cells comprise a genetic disruption at a target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, for example, to knock-out (KO) or reduce or eliminate the expression of the gene product of the TGFBR2 locus. In some aspects, the provided engineered T cells also comprise a modified T cell receptor alpha constant (TRAC) locus comprising a transgene encoding the recombinant TCR, or portion thereof, such as one or more chains of the recombinant TCR. In some aspects, the transgene (e.g., sequences that are exogenous or heterologous to the T cell) encoding the recombinant TCR or a portion thereof, is integrated at the TRAC locus of the T cell, by targeted knock-in (KI), and the expression of the endogenous TRAC gene product, the TCRa constant region, is reduced or eliminated.

[0167] In some aspects, the provided cells are engineered by CRISPR/Cas9 mediated gene editing to introduce a genetic disruption at a target site, and/or targeted integration (targeted knock-in, KI) of transgene sequences, for example encoding the recombinant TCR, at or near one of the target sites with the genetic disruption. In some aspects, the cells are engineered to comprise a genetic disruption to knockout (KO) the endogenous TGFBR2 locus. In some aspects, a genetic disruption is introduced at a target site at a TRAC locus, and in the presence of a polynucleotide comprising transgene sequences encoding a recombinant TCR or a portion thereof, the transgene sequences is integrated into a location at or near the target site with the genetic disruption, for example, by homology-directed repair (HDR). Exemplary methods for carrying out genetic disruption at the endogenous TGFBR2 or TRAC locus and/or for carrying out HDR for targeted integration of the transgene, such as at a target site at the endogenous TRAC locus are described in this disclosure. Further, the engineered T cells can be generated using other methods, for example, as described in WO2015/161276, W02015/070083,

WO20 19/070541, WO2019/ 195491, WO2019/195492, WO2019/089884, and WO2020/223535, the contents of which are incorporated by reference.

A. Genetic Disruption

[0168] In some embodiments, one or more genetic disruption is induced at one or more target sites in the T cell. In some aspects, during the engineering of the T cell, one or more genetic disruption is induced at one or more target sites in the T cell. In some aspects, at least two genetic disruption is induced, one at a target site at the endogenous TGFBR2 locus, and another at a target site at the endogenous TRAC locus. In some aspects, the genetic disruption at a target site at the endogenous TRAC locus then results in targeted integration of the transgene sequences (e.g., encoding a recombinant TCR), at or near that target site.

[0169] In some aspects, the genetic disruption at a target site at the endogenous TGFBR2 locus can be referred to as a first genetic disruption. In some aspects, a target site at the endogenous TGFBR2 locus can be referred to as a first target site. In some aspects, the genetic disruption at a target site at the endogenous TRAC locus can be referred to as a second genetic disruption. In some aspects, a target site at the endogenous TRAC locus can be referred to as a second target site.

[0170] In some embodiments, one or more targeted genetic disruption, for example a first genetic disruption, is induced at the endogenous TGFBR2 locus. In some embodiments, one or more targeted genetic disruption is induced at one or more target sites, for example a first target site, at or near the endogenous TGFBR2 locus. In some embodiments, the first genetic disruption is induced in an exon of the endogenous TGFBR2 locus. In some embodiments, the first genetic disruption is induced in an intron of the endogenous TGFBR2 locus.

[0171] In some embodiments, one or more targeted genetic disruption, for example a second genetic disruption, is induced at the endogenous TRAC locus. In some embodiments, one or more targeted genetic disruption is induced at one or more target sites, for example a second target site, at or near the endogenous TRAC locus. In some embodiments, the second genetic disruption is induced in an exon of the endogenous TRAC locus. In some embodiments, the second genetic disruption is induced in an intron of the endogenous TRAC locus. In some aspects, the presence of the one or more second genetic disruption and a polynucleotide, e.g., a template polynucleotide that contains transgene sequences encoding a recombinant TCR or a portion thereof, can result in targeted integration of the transgene sequences at or near the one or more genetic disruption (e.g., second target site) at the endogenous TRAC locus. In some aspects, such targeted integration produces a modified TRAC locus comprising a transgene encoding the recombinant TCR, or a portion of the recombinant TCR.

[0172] In some embodiments, genetic disruption results in a DNA break or a nick. In some embodiments, at the site of the DNA break, action of cellular DNA repair mechanisms can result in a knock-out (KO), an indel, an insertion, a missense or a frameshift mutation, such as a biallelic frameshift mutation, and/or a deletion of all or part of the gene. In some embodiments, the genetic disruption can be targeted to one or more exon of a gene or portion thereof, such as within the first or second exon. In some embodiments, a DNA binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the sequences at a region near one of the at least one target site(s), is used for targeted disruption. In some aspects, in the absence of exogenous template polynucleotides for HDR the disruption, the targeted genetic disruption results in an indel, a deletion, a mutation and/or an insertion within an exon of the gene.

[0173] In some embodiments, polynucleotides, e.g., template polynucleotides that include a transgene encoding a recombinant TCR or a portion thereof, and homology sequences, can be introduced for targeted integration of the recombinant TCR-encoding transgene at or near the site of the genetic disruption, for example a second target site at the TRAC locus, by HDR.

[0174] In some embodiments, the genetic disruption is carried by introducing one or more agent(s) capable of inducing a genetic disruption. In some embodiments, such agents comprise a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the gene. In some embodiments, the agent comprises various components, such as a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease. In some embodiments, the agents can target one or more target sites, e.g., a first target site at a TGFBR2 locus and/or a second target site at a TRAC locus.

[0175] In some embodiments, the genetic disruption occurs at a target site (also referred to and/or known as “target position,” “target DNA sequence,” or “target location”). In some embodiments, target site is or includes a site on a target DNA (e.g., genomic DNA) that is modified by the one or more agent(s) capable of inducing a genetic disruption, e.g., a Cas9 molecule complexed with a gRNA that specifies the target site. For example, in some embodiments, the target site may include locations in the DNA, e.g., at an endogenous TGFBR2 and/or TRAC loci, where cleavage or DNA breaks occur. In some aspects, integration of nucleic acid sequences by HDR can occur at or near the target site or target sequence. In some embodiments, a target site can be a site between two nucleotides, e.g., adjacent nucleotides, on the DNA into which one or more nucleotides is added. The target site may comprise one or more nucleotides that are altered by a template polynucleotide. In some embodiments, the target site is within a target sequence (e.g., the sequence to which the gRNA binds). In some embodiments, a target site is upstream or downstream of a target sequence.

[0176] In some embodiments, genetic disruption results in a DNA break, such as a doublestrand break (DSB) or a cleavage, or a nick, such as a single-strand break (SSB), at one or more target site in the genome. In some embodiments, at the site of the genetic disruption, e.g., DNA break or nick, action of cellular DNA repair mechanisms can result in knock-out, insertion, missense or frameshift mutation, such as a biallelic frameshift mutation, deletion of all or part of the gene; or, in the presence of a repair template, e.g., a template polynucleotide, can alter the DNA sequence based on the repair template, such as integration or insertion of the nucleic acid sequences, such as a transgene encoding all or a portion of a recombinant TCR, contained in the template. In some embodiments, the genetic disruption can be targeted to one or more exon of a gene or portion thereof. In some embodiments, the genetic disruption can be targeted near a desired site of targeted integration of exogenous sequences, e.g., transgene sequences encoding a recombinant TCR.

1. Target Site at an Endogenous TGFBR2 Locus for Gene Editing

[0177] In some aspects, the provided engineered T cells comprise a genetic disruption at a target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, for example, to knock-out (KO) or reduce or eliminate the expression of the gene product of the TGFBR2 locus, TGFBR2. In some aspects, a genetic disruption at a target site at an endogenous TGFBR2 locus is referred to as a first genetic disruption, and a target site at the endogenous TGFBR2 locus is referred to as a first target site.

[0178] In some embodiments, the genetic disruption, e.g., first genetic disruption, is targeted at an endogenous or genomic locus that encodes the transforming growth factor-beta receptor type II (also known as TGFBRII, TGFBR2, TGFR-2, TGFp-RII, TGFbeta-RII, TBR-ii, TBRII, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC or TAAD2).

[0179] In humans, TGFBR2 is encoded by the transforming growth factor-beta receptor type-2 (TGFBR2) gene. In some embodiments, the genetic disruption is targeted at the human TGFBR2 locus, via homology-directed repair (HDR). In some aspects, the genetic disruption is targeted at a target site within the TGFBR2 locus containing an open reading frame encoding TGFBR2, such that the genetic disruption occurs at or near a first target site at the TGFBR2 locus. In some aspects, the genetic disruption is targeted at or near an exon of the open reading frame encoding TGFBR2. In some aspects, the genetic disruption is targeted at or near an intron of the open reading frame encoding TGFBR2.

[0180] In some aspects, a genetic disruption of the TGFBR2 locus reduces expression of the gene product of the TGFBR2 locus in the T cells. In some embodiments, the reduced expression of TGFBR2 includes reduced expression of a TGFBR2 mRNA. In some embodiments, the genetic disruption that reduces expression of TGFBR2 includes reduced expression of TGFBR2 protein, the protein encoded by the TGFBR2 mRNA. In some embodiments, the genetic disruption eliminates TGFBR2 gene activity. In some embodiments, the genetic disruption includes inactivation or disruption of both alleles of the TGFBR2 locus. In some embodiments, the genetic disruption includes inactivation or disruption of all alleles of the TGFBR2 locus. In some embodiments, the genetic disruption comprises inactivation or disruption of all TGFBR2 coding sequences in the cell. In some embodiments, the genetic disruption comprises insertions or deletions (indel) at the TGFBR2 locus. In some embodiments, the genetic disruption comprises an indel and results in a knock-out (KO) at the TGFBR2 locus. In some embodiments, the indel can be detected or quantitated, among a population of engineered T cells, by PCR-based methods such as ddPCR. In some embodiments, the genetic disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the TGFBR2 gene. In some embodiments, the TGFBR2 gene is knocked out. In some aspects, the genetically engineered T cell does not encode a functional TGFBR2 polypeptide. In some aspects, the genetically engineered T cell does not encode a TGFBR2 polypeptide. In some aspects, the genetically engineered T cell does not encode a full length TGFBR2 polypeptide. In some aspects, the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell. In some aspects, TGFP signal transmission is reduced or eliminated in the genetically engineered T cell.

[0181] TGFBR2 a transmembrane protein that is a member of the serine/threonine protein kinase family and the TGFB receptor subfamily. TGFBR2 forms a heterodimeric complex with TGF-beta type I serine/threonine kinase receptor (TGFBRI), a non-promiscuous receptor for the transforming growth factor beta (TGFP) cytokines TGFpi, TGFP2 and TGFP3 to transduce signals from the cytokines and regulate various physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression and carcinogenesis (see, e.g., Yang et al., Trends Immunol. (2010) 31(6): 220-227; Oh et al., J Immunol. (2013) 191(8): 3973-3979; Principe et al., Cancer Res. (2016) 76(9): 2525-2539). [0182] In some aspects, TGFP is synthesized in a latent form, and is activated to permit formation of a tetrameric receptor complex with TGFP receptors TGFBRI and TGFBR2. In some aspects, the formation of the receptor complex composed of two TGFBRI and two TGFBR2 molecules symmetrically bound to the cytokine dimer results in the phosphorylation and the activation of TGFBRI by the constitutively active TGFBR2. In some cases such as the canonical SMAD-dependent TGFP-signaling pathways, activated TGFBRI phosphorylates mothers against decapentaplegic homolog 2 (SMAD2), which dissociates from the receptor and interacts with SMAD4. The SMAD2-SMAD4 complex is subsequently translocated to the nucleus where it modulates the transcription of the TGFP-regulated genes. In some aspects, TGFBR2 can also be involved in non-canonical, SMAD-independent TGFP signaling pathways.

[0183] In the context of a tumor or a cancer, TGFP can promote tumors, e.g., by dysregulation of cyclin-dependent kinase inhibitors, alteration in cytoskeletal architecture, increases in proteases and extracellular matrix formation, decreased immune surveillance and increased angiogenesis.

[0184] In some aspects, TGFP can control immune responses and maintains immune homeostasis through its impact on proliferation, differentiation and survival of multiple immune cell lineages. In some aspects, TGFpi is the primary isoform expressed in the immune system, and has a wide-ranging regulatory activity affecting multiple types of immune cells. In some contexts, such as in T cells, binding of TGFP to TGFBR2 can downregulate, inhibit or hinder T cell activation, proliferation and differentiation. TGFP also can control immune tolerance by virtue of its effect on T cells. For immune cells that can be present in the tumor microenvironment (TME), TGFP may have an adverse effect on anti-tumor immunity and significantly inhibits tumor immune surveillance. For example, transgenic mice that express a dominant-negative TGFBR2 under a T-cell- specific promoter was observed to have spontaneous T-cell differentiation and autoimmune disease (see, e.g., Gorelik et al., Nat. Rev. Immunol. (2002) 2(l):46-53). In some aspects, TGFP can directly suppresses the cytotoxic activity of cytotoxic T lymphocytes, in some cases via transcriptional repression of genes encoding multiple key molecules, such as perforin, granzymes and cytotoxins. In some aspects, TGFP regulates the clonal expansion and cytotoxic activity of CD8+ T cells, which can then result in tumor progression or tumor promotion. In some aspects, TGFP also has a significant impact on CD4+ T-cell differentiation and function, and promotes generation of regulatory T cells (Tregs) and Thl7 cells (see, e.g., Principe et al., Cancer Res. (2016) 76(9): 2525-2539). In some aspects, as TGFP in the context of a tumor promotes tumor progression and can have immunosuppressive activity, reduction, inhibition or deletion of TGFP signaling components, e.g., TGFP receptors, can enhance T cell differentiation, function and persistence.

[0185] In some aspects, TGFP is involved in various aspects of carcinogenesis. In some contexts, impaired TGFP signaling is frequently associated with cancer progression in head and neck squamous cell carcinoma (HNSCC). In some contexts, a reduction or complete loss of TGFBR2 is observed in approximately 30% of to 87% of human HNSCC. In some aspects, a loss of Smad4 (22% to 51%) and Smad2 (14% to 38%) expression has been reported in human HNSCC. In some aspects, TGFP signaling can also be involved in tumor progression by means of loss of epithelial cell adhesion, extracellular matrix remodeling, and enhanced angiogenesis, for example, resulting in promotion of epithelial to mesenchymal transition. In some cases, the level of TGFP is elevated in HNSCC samples, for example, by 1.5- to 7.5-fold increase compared with normal tissues; and TGFP levels have been observed to increase by 1.5- to 5.3- fold in 44% of tissue samples with adjacent HNSCC.

[0186] Exemplary human TGFBR2 precursor polypeptide sequence is set forth in SEQ ID NO: 151 (isoform 1; mature polypeptide includes residues 23-567 of SEQ ID NO: 151; see Uniprot Accession No. P37173-1; NCBI Reference Sequence: NP_003233.4; mRNA sequence set forth in SEQ ID NO: 153, NCBI Reference Sequence: NM_003242.5) or SEQ ID NO: 152 (isoform 2; mature polypeptide includes residues 23-592 of SEQ ID NO: 152; see Uniprot Accession No. P37173-2; NCBI Reference Sequence: NP_001020018.1; mRNA sequence set forth in SEQ ID NO: 154, NCBI Reference Sequence: NM_001024847.2). The two isoforms are produced by alternative splicing.

[0187] An exemplary mature TGFBR2 contains an extracellular region (including amino acid residues 22-166 of the human TGFBR2 precursor sequence (isoform 1) set forth in SEQ ID NO:151, or amino acid residues 22-191 of the human TGFBR2 precursor sequence (isoform 2) set forth in SEQ ID NO:152), a transmembrane region (including amino acid residues 167-187 of the human TGFBR2 precursor sequence (isoform 1) set forth in SEQ ID NO: 151, or amino acid residues 192-212 of the human TGFBR2 precursor sequence (isoform 2) set forth in SEQ ID NO: 152), and an intracellular region (including amino acid residues 188-567 of the human TGFBR2 precursor sequence (isoform 1) set forth in SEQ ID NO: 151, or amino acid residues 213-592 of the human TGFBR2 precursor sequence (isoform 2) set forth in SEQ ID NO: 152). The TGFBR2 contains a serine-threonine/tyrosine-protein kinase catalytic domain, at amino acid residues 244-544 of the human TGFBR2 precursor sequence (isoform 1) set forth in SEQ ID NO: 151 or at amino acid residues 269-569 of the human TGFBR2 precursor sequence (isoform 2) set forth in SEQ ID NO: 152. In humans, an exemplary genomic locus encoding TGFBR2, TGFBR2, comprises an open reading frame that contains 7 exons and 6 introns for the transcript variant that encodes isoform 1, or 8 exons and 7 introns for the transcript variant that encodes isoform 2.

[0188] An exemplary mRNA transcript of TGFBR2 encoding isoform 1 can span the sequence corresponding to Chromosome 3: 30,606,502-30,694,134 on the forward strand., with reference to human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 1 sets forth the coordinates of the exons and introns of the open reading frames and the untranslated regions of the transcript encoding isoform 1 of an exemplary human TGFBR2 locus.

Table 1. Coordinates of exons and introns of exemplary human TGFBR2 locus, isoform 1

(GRCh38, Chromosome 3, forward strand).

[0189] An exemplary mRNA transcript of TGFBR2 encoding isoform 2 can span the sequence corresponding to Chromosome 3: 30,606,601-30,694,142 on the forward strand., with reference to human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 2 sets forth the coordinates of the exons and introns of the open reading frames and the untranslated regions of the transcript encoding isoform 2 of an exemplary human TGFBR2 locus.

Table 2. Coordinates of exons and introns of exemplary human TGFBR2 locus, isoform 2

(GRCh38, Chromosome 3, forward strand).

[0190] In some aspects, the genetic disruption is targeted such that upon integration of the transgene encoding the recombinant TCR, the expression of the endogenous TGFBR2 gene is reduced or eliminated.

[0191] In certain embodiments, a genetic disruption is targeted at, near, or within a TGFBR2 locus. In particular embodiments, the genetic disruption is targeted at, near, or within an open reading frame of the TGFBR2 locus (such as described in Tables 1 and 2 herein). In certain embodiments, the genetic disruption is targeted at, near, or within an open reading frame that encodes a TCRa constant domain. In some embodiments, the genetic disruption is targeted at, near, or within the TGFBR2 locus (such as described in Tables 1 and 2 herein), or a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion, e.g., at or at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the TGFBR2 locus (such as described in Tables 1 and 2 herein).

[0192] In some aspects, the target site is within an exon of the open reading frame of the endogenous TGFBR2 locus. In some aspects, the target site is within an intron of the open reading frame of the TGFBR2 locus. In some aspects, the target site is within a regulatory or control element, e.g., a promoter, 5’ untranslated region (UTR) or 3’ UTR, of the TGFBR2 locus. In some embodiments, the target site is within the TGFBR2 genomic region sequence described in Tables 1 and 2 herein or any exon or intron of the TGFBR2 genomic region sequence contained therein.

[0193] In some embodiments, the target site for a genetic disruption is selected such that after integration of the transgene sequences, the cell is knocked out for, reduced and/or eliminated expression from the endogenous TGFBR2 locus.

[0194] In some embodiments, a genetic disruption is targeted within an exon of the TGFBR2 locus or open reading frame thereof. In certain embodiments, the genetic disruption is within the first exon, second exon, third exon, or forth exon of the TGFBR2 locus or open reading frame thereof. In particular embodiments, the genetic disruption is within the first exon of the TGFBR2 locus or open reading frame thereof. In some embodiments, the genetic disruption is within 500 base pairs (bp) downstream from the 5’ end of the first exon in the TGFBR2 locus or open reading frame thereof. In particular embodiments, the genetic disruption is between the 5’ nucleotide of exon 1 and upstream of the 3’ nucleotide of exon 1. In certain embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream from the 5’ end of the first exon in the TGFBR2 locus or open reading frame thereof. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream from the 5’ end of the first exon in the TGFBR2 locus or open reading frame thereof, each inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream from the 5’ end of the first exon in the TGFBR2 locus or open reading frame thereof, inclusive.

[0195] In particular embodiments, the genetic disruption is within the fourth exon of the TGFBR2 locus or the open reading frame of the transcript encoding isoform 1 of an exemplary human TGFBR2 locus (such as described in Table 1 herein). In some embodiments, the genetic disruption is within 500 base pairs (bp) downstream from the 5’ end of the fourth exon in the TGFBR2 locus or an open reading frame thereof. In particular embodiments, the genetic disruption is between the 5’ nucleotide of exon 4 and upstream of the 3’ nucleotide of exon 4. In certain embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream from the 5’ end of the fourth exon in the TGFBR2 locus or open reading frame thereof. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream from the 5’ end of the fourth exon in the TGFBR2 locus or open reading frame thereof, each inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream from the 5’ end of the fourth exon in the TGFBR2 locus or open reading frame thereof, inclusive.

[0196] In particular embodiments, the genetic disruption is targeted within the fifth exon of the TGFBR2 locus or the open reading frame of the transcript encoding isoform 2 of an exemplary human TGFBR2 locus (as described in Table 2 herein). In some embodiments, the genetic disruption is within 500 base pairs (bp) downstream from the 5’ end of the fifth exon in the TGFBR2 locus or an open reading frame thereof. In particular embodiments, the genetic disruption is between the 5’ nucleotide of exon 5 and upstream of the 3’ nucleotide of exon 5. In certain embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream from the 5’ end of the fifth exon in the TGFBR2 locus or open reading frame thereof. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream from the 5’ end of the fifth exon in the TGFBR2 locus or open reading frame thereof, each inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream from the 5’ end of the fifth exon in the TGFBR2 locus or open reading frame thereof, inclusive.

[0197] In some aspects, the target site is within an exon, such as exons corresponding to early coding regions. In some embodiments, the target site is within or in close proximity to exons corresponding to early coding region, e.g., exon 1, 2, 3, 4 or 5 of the open reading frame of the endogenous TGFBR2 locus (such as described in Tables 1 and 2 herein), or including sequence immediately following a transcription start site, within exon 1, 2, 3, 4 or 5, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, 3, 4 or 5. In some aspects, the target site is at or near exon 1 of the endogenous TGFBR2 locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1. In some embodiments, the target site is at or near exon 2 of the endogenous TGFBR2 locus, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2. In some aspects, the target site is at or near exon 3 of the endogenous TGFBR2 locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 3. In some aspects, the target site is at or near exon 4 of the endogenous TGFBR2 locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 4. In some aspects, the target site is at or near exon 5 of the endogenous TGFBR2 locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 5. In some aspects, the target site is within a regulatory or control element, e.g., a promoter, of the TGFBR2 locus.

[0198] In some aspects, the target site is placed at or near the beginning of the endogenous open reading frame sequences encoding the intracellular regions of the TGFBR2, e.g., amino acid residues 188-567 of the human TGFBR2 precursor sequence (isoform 1) set forth in SEQ ID NO: 151, or amino acid residues 213-592 of the human TGFBR2 precursor sequence (isoform 2) set forth in SEQ ID NO: 152. In some embodiments, the target site is located at or near exon 4 of the open reading frame of the transcript encoding isoform 1 of an exemplary human TGFBR2 locus (as described in Table 1 herein), or after, downstream of or 3’ of exon 4 of the open reading frame of the transcript encoding isoform 1 of an exemplary human TGFBR2 locus (as described in Table 1 herein), or at or near exon 5 of the open reading frame of the transcript encoding isoform 2 of an exemplary human TGFBR2 locus (as described in Table 2 herein), or after, downstream of or 3 ’ of exon 5 of the open reading frame of the transcript encoding isoform 2 of an exemplary human TGFBR2 locus (as described in Table 2 herein).

[0199] In certain embodiments, a genetic disruption is targeted at, near, or within a TGFBR2 locus. In particular embodiments, the genetic disruption is targeted at, near, or within an open reading frame of the TGFBR2 locus (such as described in Table 1 or 2 herein). In certain embodiments, the genetic disruption is targeted at, near, or within an open reading frame that encodes a TGFBR2. In some embodiments, the genetic disruption is targeted at, near, or within the TGFBR2 locus (such as described in Table 1 or 2 herein), or a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion, e.g., at or at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the TGFBR2 locus (such as described in Table 1 or 2 herein).

[0200] In some embodiments, a first target site at the TGFBR2 locus the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129. In some aspects, the first target site comprises SEQ ID NO:59. In some aspects, the first target site comprises

SEQ ID NO: 59. In some aspects, the first target site comprises SEQ ID NO:60. In some aspects, the first target site comprises SEQ ID NO:61. In some aspects, the first target site comprises SEQ ID NO: 62. In some aspects, the first target site comprises SEQ ID NO:63. In some aspects, the first target site comprises SEQ ID NO:64. In some aspects, the first target site comprises SEQ ID NO:65. In some aspects, the first target site comprises SEQ ID NO:66. In some aspects, the first target site comprises SEQ ID NO:67. In some aspects, the first target site comprises SEQ ID NO: 68. In some aspects, the first target site comprises SEQ ID NO:69. In some aspects, the first target site comprises SEQ ID NO:70. In some aspects, the first target site comprises SEQ ID NO: 71. In some aspects, the first target site comprises SEQ ID NO:72. In some aspects, the first target site comprises SEQ ID NO:73. In some aspects, the first target site comprises SEQ ID NO: 74. In some aspects, the first target site comprises SEQ ID NO:75. In some aspects, the first target site comprises SEQ ID NO:76. In some aspects, the first target site comprises SEQ ID NO: 77. In some aspects, the first target site comprises SEQ ID NO:78. In some aspects, the first target site comprises SEQ ID NO:79. In some aspects, the first target site comprises SEQ ID NO:80. In some aspects, the first target site comprises SEQ ID NO:81. In some aspects, the first target site comprises SEQ ID NO:82. In some aspects, the first target site comprises SEQ ID NO: 83. In some aspects, the first target site comprises SEQ ID NO:84. In some aspects, the first target site comprises SEQ ID NO:85. In some aspects, the first target site comprises

SEQ ID NO:86. In some aspects, the first target site comprises SEQ ID NO:87. In some aspects, the first target site comprises SEQ ID NO:88. In some aspects, the first target site comprises SEQ ID NO:89. In some aspects, the first target site comprises SEQ ID NO:90. In some aspects, the first target site comprises SEQ ID NO:91. In some aspects, the first target site comprises SEQ ID NO:92. In some aspects, the first target site comprises SEQ ID NO:93. In some aspects, the first target site comprises SEQ ID NO:94. In some aspects, the first target site comprises SEQ ID NO:95. In some aspects, the first target site comprises SEQ ID NO:96. In some aspects, the first target site comprises SEQ ID NO:97. In some aspects, the first target site comprises SEQ ID NO:98. In some aspects, the first target site comprises SEQ ID NO:99. In some aspects, the first target site comprises SEQ ID NO: 100. In some aspects, the first target site comprises SEQ ID NO: 101. In some aspects, the first target site comprises SEQ ID NO: 102. In some aspects, the first target site comprises SEQ ID NO: 103. In some aspects, the first target site comprises SEQ ID NO: 104. In some aspects, the first target site comprises SEQ ID NO: 105. In some aspects, the first target site comprises SEQ ID NO: 106. In some aspects, the first target site comprises SEQ ID NO: 107. In some aspects, the first target site comprises SEQ ID NO: 108. In some aspects, the first target site comprises SEQ ID NO: 109. In some aspects, the first target site comprises SEQ ID NO: 110. In some aspects, the first target site comprises SEQ ID NO: 111. In some aspects, the first target site comprises SEQ ID NO: 112. In some aspects, the first target site comprises SEQ ID NO: 113. In some aspects, the first target site comprises SEQ ID NO: 114. In some aspects, the first target site comprises SEQ ID NO: 115. In some aspects, the first target site comprises SEQ ID NO: 116. In some aspects, the first target site comprises SEQ ID NO: 117. In some aspects, the first target site comprises SEQ ID NO: 118. In some aspects, the first target site comprises SEQ ID NO: 119. In some aspects, the first target site comprises SEQ ID NO: 120. In some aspects, the first target site comprises SEQ ID NO: 121. In some aspects, the first target site comprises SEQ ID NO: 122. In some aspects, the first target site comprises SEQ ID NO: 123. In some aspects, the first target site comprises SEQ ID NO: 124. In some aspects, the first target site comprises SEQ ID NO: 125. In some aspects, the first target site comprises SEQ ID NO: 126. In some aspects, the first target site comprises SEQ ID NO: 127. In some aspects, the first target site comprises SEQ ID NO: 128. In some aspects, the first target site comprises SEQ ID NO: 129.

[0201] In some embodiments, for genetic disruption using a CRISPR/Cas based gene editing, a gRNA sequences that is or comprises a targeting domain sequence (in some cases also referred to as a spacer sequence) that can bind to and/or target a target site in the genome, e.g., a first target site at a TGFBR2 locus. A genome- wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) sequences targeting constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target site or position.

[0202] In some embodiments, the target site target domain is at or near the TGFBR2 locus. In some aspects, the target site, such as the first target site, is selected from any one of SEQ ID NOS:59-129. In some embodiments, the target site that the targeting domain of the gRNA binds to or targets is located at an early coding region of a gene of interest, such as TGFBR2. Targeting of the early coding region can be used to genetic disruption (i.e., eliminate expression of) the gene of interest. In some embodiments, the early coding region of a gene of interest includes sequence immediately following a start codon (e.g., ATG), or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40bp, 30bp, 20bp, or lObp). In particular examples, the target nucleic acid is within 200bp, 150bp, 100 bp, 50 bp, 40bp, 30bp, 20bp or lObp of the start codon. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target site or a complement of the target site, such as the target nucleic acid in the TGFBR2 locus, or the targeting domain of the gRNA can bind to or hybridize to the target site or a complement of the target site.

[0203] In some embodiments, the gRNA can target a site at the TGFBR2 locus near a desired site of targeted integration of transgene sequences, e.g., encoding a recombinant receptor. In some aspects, the gRNA can target a site based on the amount of sequences encoding the TGFBR2 that is desired for expression in the cell expressing the recombinant receptor. In some aspects, the gRNA can target a site within an exon of the open reading frame of the endogenous TGFBR2 locus. In some aspects, the gRNA can target a site within an intron of the open reading frame of the TGFBR2 locus. In some aspects, the gRNA can target a site within a regulatory or control element, e.g., a promoter, of the TGFBR2 locus. In some aspects, the target site at the TGFBR2 locus that is targeted by the gRNA can be any target sites described herein, e.g., in Section I.A.l. In some embodiments, the gRNA can target a site within or in close proximity to exons corresponding to early coding region, e.g., exon 1, 2, 3, 4 or 5 of the open reading frame of the endogenous TGFBR2 locus, or including sequence immediately following a transcription start site, within exon 1, 2, 3, 4 or 5, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, 3, 4 or 5. In some embodiments, the gRNA can target a site at or near exon 2 of the endogenous TGFBR2 locus, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2.

[0204] In some aspects, a first genetic disruption is introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some aspects, the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein. In some aspects, exemplary gRNAs (e.g., exemplary first gRNAs) that target a target site at the endogenous TRBC2 locus include a sequence of ribonucleic acids (e.g., targeting domain sequences) that can bind to or target or is complementary to or can bind to the complimentary strand sequence of the target site set forth in any one of SEQ ID NOS:59-129. Any of the known methods can be used to target and generate a genetic disruption of the endogenous TGFBR2 locus can be used in the embodiments provided herein. Exemplary first gRNA targeting domain sequences (e.g., which target a first target site at the TGFBR2 locus) include a sequence selected from any one of SEQ ID NOS: 58, and ISO- 135. In some aspects, the first gRNA targeting domain comprises SEQ ID NO:58. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 130. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 131. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 132. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 133. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 134. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 135.

2. Target Site at an Endogenous TRAC Locus for Integration by HDR [0205] In some embodiments, the genetic disruption occurs at the endogenous genes that encode one or more domains, regions and/or chains of the endogenous T cell receptor (TCR). In some embodiments, the genetic disruption is targeted at the endogenous gene locus that encodes TCRa. In some embodiments, the genetic disruption is targeted at the gene encoding TCRa constant domain (TRAC in humans). In some aspects, a genetic disruption at a target site at an endogenous TRAC locus is referred to as a second genetic disruption, and a target site at the endogenous TRAC locus is referred to as a second target site.

[0206] In some aspects, in the presence of a genetic disruption at a target site (e.g., second target site) at a TRAC locus, and a polynucleotide, such as the template polynucleotide having homology with sequences at or near the target site in an endogenous TRAC locus can be used to alter the structure of a target DNA, e.g., targeted insertion (for example, a knock-in (KI)) of the transgene encoding a recombinant TCR or a portion thereof at or around the TRAC locus, for example by homology-dependent repair (HDR), which is described further herein, for example, in Section I.B. In some embodiments, the homology sequences of the template polynucleotide target the transgene at a TRAC locus. [0207] In some aspects, a genetic disruption and targeted insertion (e.g., a knock-in) of the transgene encoding a recombinant TCR or a portion thereof, at the TRAC locus reduces expression of the gene product of the TRAC locus (e.g., endogenous TCR alpha chain constant region (Ca)) in the T cells. In some embodiments, the reduced expression of TRAC includes reduced expression of an endogenous TRAC mRNA. In some embodiments, the genetic disruption that reduces expression of TRAC includes reduced expression of an endogenous TCR alpha chain constant region (Ca) protein, the protein encoded by the TRAC mRNA. In some embodiments, the genetic disruption eliminates TRAC gene activity. In some embodiments, the genetic disruption includes inactivation or disruption of both alleles of the TRAC locus. In some embodiments, the genetic disruption includes inactivation or disruption of all alleles of the TRAC locus. In some embodiments, the genetic disruption comprises inactivation or disruption of all TRAC coding sequences in the cell. In some embodiments, the genetic disruption comprises an insertion of the transgene at the TRAC locus. In some embodiments, the genetic disruption comprises an indel at the TRAC locus. In some embodiments, the genetic disruption comprises an indel and results in a knock-out (KO) at the TRAC locus. In some embodiments, the TRAC indels can be detected or quantitated, among a population of engineered T cells, by PCR-based methods such as ddPCR. In some aspects, the PCR-based method is designed to specifically recognize the wild-type TRAC sequence, and not the recombinant TCR, since the recombinant TCR is inserted into the edited endogenous TRAC locus. In some embodiments, the genetic disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the TRAC gene. In some embodiments, the TRAC gene is knocked out. In some aspects, the genetically engineered T cell does not encode a functional endogenous Ca polypeptide. In some aspects, the genetically engineered T cell does not encode an endogenous Ca polypeptide. In some aspects, the genetically engineered T cell does not encode a full length endogenous Ca polypeptide. In some aspects, the expression of an endogenous Ca polypeptide is reduced or eliminated in the genetically engineered T cell. In some aspects, the pairing of a TCRP chain with a TCRa chain comprising an endogenous Ca is reduced or eliminated in the genetically engineered T cell.

[0208] In some embodiments, targeted genetic disruption of an endogenous TCR gene locus, e.g., TRAC, can lead to a reduced risk or chance of mispairing between chains of the engineered or recombinant TCR and the endogenous TCR. Mispaired TCRs can, in some aspects, create a new TCR that could potentially result in a higher risk of undesired or unintended antigen recognition and/or side effects, and/or could reduce expression levels of the desired engineered or recombinant TCR. In some aspects, reducing or preventing endogenous TCR expression can increase expression of the engineered or recombinant TCR in the T cells or T cell compositions as compared to cells in which expression of the TCR is not reduced or prevented. In some embodiments, recombinant TCR expression can be increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5 -fold or more.

[0209] In some embodiments, the endogenous TCR Ca is encoded by the TRAC gene (IMGT nomenclature). An exemplary sequence of the human T cell receptor alpha chain constant domain (TRAC) gene locus is set forth in SEQ ID NO:278 (NCBI Reference Sequence: NG_001332.3, TRAC). In some embodiments, the encoded endogenous Ca comprises the sequence of amino acids set forth in SEQ ID NO: 9, 167-172, 175, 176, and 178-181. In certain embodiments, a genetic disruption, e.g., a second genetic disruption, is targeted at, near, or within a TRAC locus. In particular embodiments, the genetic disruption is targeted at, near, or within an open reading frame of the TRAC locus. In certain embodiments, the genetic disruption is targeted at, near, or within an open reading frame that encodes a TCRa constant domain. In some embodiments, the genetic disruption is targeted at, near, or within a locus having the nucleic acid sequence set forth in SEQ ID NO:278, or a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion, e.g., at or at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the nucleic acid sequence set forth in SEQ ID NO:278.

[0210] In humans, an exemplary genomic locus of TRAC comprises an open reading frame that contains 4 exons and 3 introns. An exemplary mRNA transcript of TRAC can span the sequence corresponding to coordinates Chromosome 14: 22,547,506-22,552,154, on the forward strand, with reference to human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 3 sets forth the coordinates of the exons and introns of the open reading frames and the untranslated regions of the transcript of an exemplary human TRAC locus.

Table 3. Coordinates of exons and introns of exemplary human TRAC locus (GRCh38, Chromosome 14, forward strand). [0211] In some aspects, the transgene (e.g., exogenous nucleic acid sequences) within the template polynucleotide can be used to guide the location of target sites and/or homology arms. In some aspects, the target site of genetic disruption can be used as a guide to design template polynucleotides and/or homology arms used for HDR. In some embodiments, the genetic disruption can be targeted near a desired site of targeted integration of transgene sequences (e.g., encoding a recombinant TCR or a portion thereof). In some aspects, the target site is within an exon of the open reading frame of the TRAC locus. In some aspects, the target site is within an intron of the open reading frame of the TRAC locus.

[0212] In some embodiments, the genetic disruption is targeted at or in close proximity to the beginning of the coding region (e.g., the early coding region, e.g., within 500bp from the start codon or the remaining coding sequence, e.g., downstream of the first 500bp from the start codon). In some embodiments, the genetic disruption is targeted at early coding region of a gene of interest, e.g., TRAC, including sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp), or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).

[0213] In some embodiments, the target site is within an exon of the endogenous TRAC locus. In certain embodiments, the target site is within an intron of the endogenous TRAC locus. In some aspects, the target site is within a regulatory or control element, e.g., a promoter, 5’ untranslated region (UTR) or 3’ UTR, of the TRAC locus. In certain embodiments, the target site is within an open reading frame of an endogenous TRAC locus. In particular embodiments, the target site is within an exon within the open reading frame of the TRAC locus.

[0214] In particular embodiments, the genetic disruption is targeted at or within an open reading frame of a gene or locus of interest, e.g., TRAC locus. In some embodiments, the genetic disruption is targeted at or within an intron within the open reading frame of a gene or locus of interest. In some embodiments, the genetic disruption is targeted within an exon within the open reading frame of the gene or locus of interest.

[0215] In particular embodiments, a genetic disruption is targeted at or within an intron. In certain embodiments, a genetic disruption is targeted at or within an exon. In some embodiments, a genetic disruption is targeted at or within an exon of a gene of interest, e.g., TRAC locus.

[0216] In some embodiments, a genetic disruption is targeted within an exon of the TRAC gene, open reading frame, or locus. In certain embodiments, the genetic disruption is within the first exon, second exon, third exon, or fourth exon of the TRAC gene, open reading frame, or locus. In particular embodiments, the genetic disruption is within the first exon of the TRAC gene, open reading frame, or locus. In some embodiments, the genetic disruption is within 500 base pairs (bp) downstream from the 5’ end of the first exon in the TRAC gene, open reading frame, or locus. In particular embodiments, the genetic disruption is between the most 5’ nucleotide of exon 1 and upstream of the most 3’ nucleotide of exon 1. In certain embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream from the 5’ end of the first exon in the TRAC gene, open reading frame, or locus. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream from the 5’ end of the first exon in the TRAC gene, open reading frame, or locus, each inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream from the 5’ end of the first exon in the TRAC gene, open reading frame, or locus, inclusive.

[0217] In some aspects, the target site is within an exon, such as exons corresponding to early coding regions. In some embodiments, the target site is within or in close proximity to exons corresponding to early coding region, e.g., exon 1, 2, 3, 4 or 5 of the open reading frame of the endogenous TRAC locus (such as described in Table 3 herein), or including sequence immediately following a transcription start site, within exon 1, 2, 3, 4 or 5, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, 3, 4 or 5. In some aspects, the target site is at or near exon 1 of the endogenous TRAC locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1. In some embodiments, the target site is at or near exon 2 of the endogenous TRAC locus, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2. In some aspects, the target site is at or near exon 3 of the endogenous TRAC locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 3. In some aspects, the target site is at or near exon 4 of the endogenous TRAC locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 4. In some aspects, the target site is at or near exon 5 of the endogenous TRAC locus, e.g., within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 5. In some aspects, the target site is within a regulatory or control element, e.g., a promoter, of the TRAC locus.

[0218] In certain embodiments, a genetic disruption is targeted at, near, or within a TRAC locus. In particular embodiments, the genetic disruption is targeted at, near, or within an open reading frame of the TRAC locus (such as described in Table 3 herein). In certain embodiments, the genetic disruption is targeted at, near, or within an open reading frame that encodes a TRAC. In some embodiments, the genetic disruption is targeted at, near, or within the TRAC locus (such as described in Table 3 herein), or a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion, e.g., at or at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the TRAC locus (such as described in Table 3 herein).

[0219] In some embodiments, a second target site at the TRAC locus the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265. In some aspects, the second target site comprises SEQ ID NO:235. In some aspects, the second target site comprises SEQ ID NO:236. In some aspects, the second target site comprises SEQ ID NO:237. In some aspects, the second target site comprises SEQ ID NO:238. In some aspects, the second target site comprises SEQ ID NO:239. In some aspects, the second target site comprises SEQ ID NO:240. In some aspects, the second target site comprises SEQ ID NO:241. In some aspects, the second target site comprises SEQ ID NO:242. In some aspects, the second target site comprises SEQ ID NO:243. In some aspects, the second target site comprises SEQ ID NO:244. In some aspects, the second target site comprises SEQ ID NO:245. In some aspects, the second target site comprises SEQ ID NO:246. In some aspects, the second target site comprises SEQ ID NO:247. In some aspects, the second target site comprises SEQ ID NO:248. In some aspects, the second target site comprises SEQ ID NO:249. In some aspects, the second target site comprises SEQ ID NO:250. In some aspects, the second target site comprises SEQ ID NO:251. In some aspects, the second target site comprises SEQ ID NO:252. In some aspects, the second target site comprises SEQ ID NO:253. In some aspects, the second target site comprises SEQ ID NO:254. In some aspects, the second target site comprises SEQ ID NO:255. In some aspects, the second target site comprises SEQ ID NO:256. In some aspects, the second target site comprises SEQ ID NO:257. In some aspects, the second target site comprises SEQ ID NO:258. In some aspects, the second target site comprises SEQ ID NO:259. In some aspects, the second target site comprises SEQ ID NO:260. In some aspects, the second target site comprises SEQ ID NO:261. In some aspects, the second target site comprises SEQ ID NO:262. In some aspects, the second target site comprises SEQ ID NO:263. In some aspects, the second target site comprises SEQ ID NO:264. In some aspects, the second target site comprises SEQ ID NO:265. In some aspects, the second target site comprises SEQ ID NO:266.

[0220] In some embodiments, for genetic disruption using a CRISPR/Cas based gene editing, a gRNA sequences that is or comprises a targeting domain sequence (in some cases also referred to as a spacer sequence) that can bind to and/or target a target site in the genome, e.g., a second target site at a TRAC locus. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) sequences targeting constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target site or position.

[0221] In some embodiments, the target site target domain is at or near the TRAC locus. In some aspects, the target site, such as the second target site, is selected from any one of SEQ ID NOS:235-265. In some embodiments, the target site that the targeting domain of the gRNA binds to or targets is located at an early coding region of a gene of interest, such as TRAC. Targeting of the early coding region can be used to genetic disruption (i.e., eliminate expression of) the gene of interest. In some embodiments, the early coding region of a gene of interest includes sequence immediately following a start codon (e.g., ATG), or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40bp, 30bp, 20bp, or lObp). In particular examples, the target nucleic acid is within 200bp, 150bp, 100 bp, 50 bp, 40bp, 30bp, 20bp or lObp of the start codon. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target site or a complement of the target site, such as the target nucleic acid in the TRAC locus, or the targeting domain of the gRNA can bind to or hybridize to the target site or a complement of the target site.

[0222] In some embodiments, the gRNA can target a site at the TRAC locus near a desired site of targeted integration of transgene sequences, e.g., encoding a recombinant receptor. In some aspects, the gRNA can target a site based on the amount of sequences encoding the TRAC that is desired for expression in the cell expressing the recombinant receptor. In some aspects, the gRNA can target a site such that upon integration of the transgene sequences, e.g., encoding a recombinant receptor, the resulting TRAC locus encodes a dominant negative form of the TGFBR2. In some aspects, the gRNA can target a site within an exon of the open reading frame of the endogenous TRAC locus. In some aspects, the gRNA can target a site within an intron of the open reading frame of the TRAC locus. In some aspects, the gRNA can target a site within a regulatory or control element, e.g., a promoter, of the TRAC locus. In some aspects, the target site at the TRAC locus that is targeted by the gRNA can be any target sites described herein, e.g., in Section I.A.2. In some embodiments, the gRNA can target a site within or in close proximity to exons corresponding to early coding region, e.g., exon 1, 2, 3, 4 or 5 of the open reading frame of the endogenous TRAC locus, or including sequence immediately following a transcription start site, within exon 1, 2, 3, 4 or 5, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, 3, 4 or 5. In some embodiments, the gRNA can target a site at or near exon 2 of the endogenous TRAC locus, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2.

[0223] In some aspects, a second genetic disruption is introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some aspects, the second agent comprises a second CRISPR-Cas combination comprising a second guide RNA (gRNA) comprising a second targeting domain that binds to the second target site, and a Cas9 protein. In some aspects, exemplary gRNAs (e.g., exemplary second gRNAs) that target a target site at the endogenous TRBC2 locus include a sequence of ribonucleic acids (e.g., targeting domain sequences) that can bind to or target or is complementary to or can bind to the complimentary strand sequence of the target site set forth in any one of SEQ ID NOS:235-265. Any of the known methods can be used to target and generate a genetic disruption of the endogenous TRAC locus can be used in the embodiments provided herein. Exemplary second gRNA targeting domain sequences (e.g., which target a second target site at the TRAC locus) include a sequence selected from any one of SEQ ID NOS: 25-55. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:25. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:26. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:27. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:28. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:29. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:30. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:31. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:32. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:33. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:34. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:35. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:36. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:37. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:38. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:39. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:40. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:41. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:42. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:43. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:44. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:45. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:46. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:47. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:48. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:49. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:50. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:51. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:52. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:53. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:54. In some aspects, the second gRNA targeting domain comprises SEQ ID NO:55.

3. Methods for Genetic Disruption

[0224] In some aspects, the methods for generating the genetically engineered cells involve introducing a genetic disruption at one or more target site(s), e.g., one or more target sites at a TGFBR2 and/or TRAC locus.

[0225] Methods for generating a genetic disruption, including those described herein, can involve the use of one or more agent(s) capable of inducing a genetic disruption, such as engineered systems to induce a genetic disruption, a cleavage and/or a double strand break (DSB) or a nick in a target site or target position in the endogenous DNA such that repair of the break by an error bom process such as non-homologous end joining (NHEJ) or repair using a repair template HDR can result in the knock out of a gene and/or the insertion of a sequence of interest (e.g., exogenous nucleic acid sequences or transgene encoding a portion of a chimeric receptor) at or near the target site or position. Also provided are one or more agent(s) capable of inducing a genetic disruption, for example at one or more target sites described herein, for use in the methods provided herein. In some aspects, the one or more agent(s) can be used in combination with the template nucleotides provided herein, for homology directed repair (HDR) mediated targeted integration of the transgene sequences. Also provided are polynucleotides (e.g., nucleic acid molecules) encoding one or more components of the one or more agent(s) capable of inducing a genetic disruption.

[0226] In some aspects, the methods for generating the genetically engineered cells involve introducing a genetic disruption at a first target site at a TGFBR2 locus and/or a second target site at a TRAC locus.

[0227] In some aspects, the first genetic disruption is introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some aspects, the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein. In some aspects, the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein. [0228] In some aspects, the second genetic disruption has been introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination. In some aspects, the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein. In some aspects, the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

[0229] In some embodiments, the one or more agent(s) specifically targets the at least one target site(s), e.g., a first target site at a TGFBR2 locus and/or a second target site at a TRAC locus. In some embodiments, the agent comprises a ZFN, TAEEN or a CRISPR/Cas9 combination that specifically binds to, recognizes, or hybridizes to the target site(s). In some embodiments, the CRISPR/Cas9 system includes an engineered crRNA/tracr RNA (“single guide RNA”) to guide specific cleavage. In some embodiments, the agent comprises nucleases based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, (Swarts et al. (2014) Nature 507(7491): 258-261). Targeted cleavage using any of the nuclease systems described herein can be exploited to insert the sequences of a transgene, e.g., nucleic acid sequences encoding a recombinant TCR, into a specific target location, e.g., at a TRAC locus, using either HDR or NHEJ-mediated processes.

[0230] In some embodiments, the one or more agent(s) capable of inducing a genetic disruption comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to a particular site or position in the genome, e.g., a target site or target position. In some aspects, the targeted genetic disruption, e.g., DNA break or cleavage, of the endogenous genes encoding TCR or TGFBR2 is achieved using a protein or a nucleic acid is coupled to or complexed with a gene editing nuclease, such as in a chimeric or fusion protein. In some embodiments, the one or more agent(s) capable of inducing a genetic disruption comprises an RNA-guided nuclease, or a fusion protein comprising a DNA-targeting protein and a nuclease.

[0231] In some embodiments, the agent comprises various components, such as an RNA- guided nuclease, or a fusion protein comprising a DNA-targeting protein and a nuclease. In some embodiments, the targeted genetic disruption is carried out using a DNA-targeting molecule that includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like effectors (TALEs), fused to a nuclease, such as an endonuclease. In some embodiments, the targeted genetic disruption is carried out using RNA-guided nucleases such as a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) system (including Cas9). In some embodiments, the targeted genetic disruption is carried using agents capable of inducing a genetic disruption, such as sequence- specific or targeted nucleases, including DNA-binding targeted nucleases and gene editing nucleases such as zinc finger nucleases (ZFN) and transcription activator- like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas) system, specifically designed to be targeted to the at least one target site(s), sequence of a gene or a portion thereof. Exemplary ZFNs, TALEs, and TALENs are described in, e.g., Lloyd et al., Frontiers in Immunology, 4(221): 1-7 (2013).

[0232] Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, e.g., U.S. Pat. Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130196373;

20140120622; 20150056705; 20150335708; 20160030477 and 20160024474, the disclosures of which are incorporated by reference in their entireties.

[0233] A designed protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

[0234] Zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring ZFP or TALE protein. Engineered DNA binding proteins (ZFPs or TALEs) are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, e.g., U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 20110301073. [0235] In some cases, the DNA-targeting molecule is or comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN). For example, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some cases, the cleavage domain is from the Type IIS restriction endonuclease FokI, which generally catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, e.g., U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269: 978-982. Some gene-specific engineered zinc fingers are available commercially. For example, a platform called CompoZr, for zinc-finger construction is available that provides specifically targeted zinc fingers for thousands of targets. See, e.g., Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some cases, commercially available zinc fingers are used or are custom designed.

[0236] In some embodiments, the one or more target site(s), e.g., at a second target site at the TRAC locus genes can be targeted for genetic disruption by engineered ZFNs. Exemplary ZFN that target endogenous T cell receptor (TCR) genes include those described in, e.g., US 2015/0164954, US 2011/0158957, US 2015/0056705, US 8956828 and Torikawa et al. (2012) Blood 119:5697-5705, the disclosures of which are incorporated by reference in their entireties.

[0237] Transcription Activator like Effector (TALE) are proteins from the bacterial species Xanthomonas comprise a plurality of repeated sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from different bacterial species. In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.

[0238] In some embodiments, a “TALE-nuclease” (TALEN) is a fusion protein comprising a nucleic acid binding domain typically derived from a Transcription Activator Like Effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain comprises a nuclease domain or a domain having endonuclease activity, like for instance I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease like for instance LCrel and LOnuI or functional variant thereof. In some embodiments, the TALEN is a monomeric TALEN. A monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927. TALENs have been described and used for gene targeting and gene modifications (see, e.g., Boch et al. (2009) Science 326(5959): 1509-12.; Moscou and Bogdanove (2009) Science 326(5959): 1501; Christian et al. (2010) Genetics 186(2): 757-61; Li et al. (2011) Nucleic Acids Res 39(1): 359-72). In some embodiments, the TGFBR2 and/or TRAC genes can be targeted for genetic disruption by engineered TALENs. Exemplary TALEN that target endogenous T cell receptor (TCR) genes include those described in, e.g., WO 2017/070429, WO 2015/136001, US20170016025 and US20150203817, the disclosures of which are incorporated by reference in their entireties.

[0239] In some embodiments, a “TtAgo” is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo is derived from the bacteria Thermus thermophilus. See, e.g. Swarts et al, (2014) Nature 507(7491): 258-261, Sheng et al., (2013) Proc. Natl. Acad. Sci. U.S.A. I l l, 652). A “TtAgo system” is all the components required including e.g. guide DNAs for cleavage by a TtAgo enzyme.

[0240] In some embodiments, an engineered zinc finger protein, TALE protein or CRISPR/Cas system is not found in nature and whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., US 5,789,538; US 5,925,523; US 6,007,988; US 6,013,453; US 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

[0241] In some embodiments, the targeted genetic disruption of the endogenous genes encoding TCR, such as TRAC in humans is carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung, (2014) Nature Biotechnology, 32(4): 347-355.

[0242] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a targeting domain sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

[0243] In some aspects, the CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding guide RNA (gRNA), which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality.

[0244] In some embodiments, the one or more agent(s) comprises a guide RNA (gRNA), such as a first gRNA, having a first targeting domain that binds to and/or is complementary with a first target site at a TGFBR2 gene or a complement thereof. In some embodiments, the one or more agent(s) comprises a guide RNA (gRNA), such as a second gRNA, having a second targeting domain that binds to and/or is complementary with a second target site at a TRAC gene or a complement thereof.

[0245] In some aspects, a “gRNA molecule” is to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as “chimeric” gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). In general, a guide sequence, e.g., guide RNA, is any polynucleotide sequences comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as the TGFBR2 and/or TRAC genes in humans, to hybridize with the target sequence at the target site and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a domain, e.g., targeting domain, of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Generally, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0246] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g. at the TGFBR2 and/or TRAC loci in humans) is used to RNA-guided nucleases, e.g., Cas, to induce a DNA break at the target site or target position. Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., WO2015/161276, W02017/193107, WO2017/093969, US2016/272999 and US2015/056705, the contents of which are incorporated by reference. Methods for introducing a genetic disruption at one or more target sites (e.g., a first target site at a TGFBR2 locus and/or a second target site at a TRAC locus) and gRNAs that target the target sites include those described in, e.g., WO2015/161276, WO2015/070083, WO2019/070541, WO2019/195491, WO2019/195492, WO2019/089884, and WO2020/223535, the contents of which are incorporated by reference.

[0247] Several exemplary gRNA structures, with domains indicated thereon, are described in WO2015/161276. While not wishing to be bound by theory, with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in WO2015/161276.

[0248] In some cases, the gRNA is a unimolecular or chimeric gRNA comprising, from 5’ to 3’: a targeting domain which targets a target site (e.g., at the TGFBR2 locus or TRAC locus); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.

[0249] In other cases, the gRNA is a modular gRNA comprising first and second strands. In these cases, the first strand preferably includes, from 5’ to 3’: a targeting domain (which targets a target site (e.g., at the TGFBR2 locus or TRAC locus); and a first complementarity domain. The second strand generally includes, from 5’ to 3’: optionally, a 5’ extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.

[0250] Examples of the placement of targeting domains include those described in WO2015/161276. The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The strand of the target nucleic acid comprising the target sequence is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature 2014 (doi: 10.1038/naturel3011). In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target site, such as the target site at a TGFBR2 locus or a TRAC locus.

[0251] The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In some embodiments, the target domain itself comprises in the 5’ to 3’ direction, an optional secondary domain, and a core domain. In some embodiments, the core domain is fully complementary with the target sequence. In some embodiments, the targeting domain is 5 to 50 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., to render it less susceptible to degradation, improve bio-compatibility, etc. By way of non-limiting example, the backbone of the target domain can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the targeting domain can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0252] In various embodiments, the targeting domain is 16-26 nucleotides in length (i.e. it is 16 nucleotides in length, or 17 nucleotides in length, or 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

[0253] Exemplary targeting domains contained within the gRNA for targeting a genetic disruption, e.g., a first genetic disruption, at the TGFBR2 locus include those described in

[0254] Exemplary targeting domains contained within the gRNA for targeting the genetic disruption of the human TGFBR2 include those described in, e.g., WO2019/089884 and WO2020/223535, or a targeting domain that can bind to the targeting sequences described in the foregoing. Exemplary targeting domains contained within the gRNA for targeting the genetic disruption of the human TGFBR2 locus include a sequence of ribonucleic acids (e.g., targeting domain sequences) that can bind to or target or is complementary to or can bind to the complimentary strand sequence of the target site set forth in any one of SEQ ID NOS:59-129. Any of the known methods can be used to target and generate a genetic disruption of the endogenous TGFBR2 locus can be used in the embodiments provided herein. Exemplary first gRNA targeting domain sequences (e.g., which target a first target site at the TGFBR2 locus) include a sequence selected from any one of SEQ ID NOS: 58, and 130-135. In some aspects, the first gRNA targeting domain comprises SEQ ID NO:58. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 130. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 131. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 132. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 133. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 134. In some aspects, the first gRNA targeting domain comprises SEQ ID NO: 135. In some embodiments, the first target site at the TGFBR2 locus comprises the sequence of SEQ ID NO:83. In some embodiments, first targeting domain for targeting the TGFBR2 locus comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAAC AG) .

[0255] Exemplary targeting domains contained within the gRNA for targeting the genetic disruption of the human TRAC include those described in, e.g., WO2015/161276, W02017/193107, WO2017/093969, US2016/272999 and US2015/056705 or a targeting domain that can bind to the targeting sequences described in the foregoing. Exemplary targeting domains contained within the gRNA for targeting the genetic disruption of the human TRAC locus using S. pyogenes or S. aureus Cas9 can include any of those set forth in Table 4. In some embodiments, the second target site at the TRAC locus comprises the sequence of SEQ ID NO:238. In some embodiments, the second targeting domain for targeting the TRAC locus comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

Table 4. Exemplary TRAC gRNA targeting domain sequences

[0256] In some embodiments, the gRNA for targeting a TGFBR2 locus or a TRAC locus can be any that are described herein, or are described elsewhere e.g., in WO2015/161276, W02017/193107, WO2017/093969, US2016/272999, US2015/056705, WO2015/070083, WO20 19/070541, WO2019/ 195491, WO2019/195492, WO2019/089884, and WO2020/223535, or a targeting domain that can bind to the targeting sequences described in the foregoing. Exemplary methods for gene editing of an endogenous locus of the T cell include those described in, e.g. US2011/0158957, US2014/0301990, US2015/0098954,US2016/0208243; US2016/272999 and US2015/056705; WO2014/191128, W02015/136001, WO2015/161276, WO20 16/069283, WO2016/016341, W02017/193107, and WO2017/093969; and Osborn et al. (2016) Mol. Ther. 24(3):570-581. Any of the known methods can be used to generate a genetic disruption of an endogenous locus of the T cell can be used in the embodiments provided herein.

[0257] In some aspects, the gRNA can target a site within an exon of the open reading frame of an endogenous TGFBR2 locus or a TRAC locus. In some aspects, the gRNA can target a site within an intron of the open reading frame of a TGFBR2 locus or a TRAC locus. In some aspects, the gRNA can target a site within a regulatory or control element, e.g., a promoter, of a TGFBR2 locus or a TRAC locus. In some aspects, the target site at a TGFBR2 locus or a TRAC locus that is targeted by the gRNA can be any target sites described herein, e.g., in Sections I.A.l or I.A.2, respectively. In some embodiments, the gRNA can target a site within or in close proximity to exons corresponding to early coding region, e.g., exon 1, 2 or 3 of the open reading frame of an endogenous TGFBR2 locus or a TRAC locus, or including sequence immediately following a transcription start site, within exon 1, 2, or 3, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, or 3. In some embodiments, the gRNA can target a site at or near exon 2 of an endogenous TGFBR2 locus or a TRAC locus, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2. [0258] In some embodiments, gRNA sequences that is or comprises a targeting domain sequence targeting the target site in a particular gene, such as a TGFBR2 or a TRAC locus, designed or identified. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) sequences targeting constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target site or position.

[0259] In some embodiments, targeting domains include those for introducing a genetic disruption at a TGFBR2 locus or a TRAC locus using S. pyogenes Cas9 or using N. meningitidis Cas9. In some embodiments, targeting domains include those for introducing a genetic disruption at a TGFBR2 locus or a TRAC locus using S. pyogenes Cas9. Any of the targeting domains can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase). In some embodiments, dual targeting is used to create two nicks on opposite DNA strands by using S. pyogenes Cas9 nickases with two targeting domains that are complementary to opposite DNA strands, e.g., a gRNA comprising any minus strand targeting domain may be paired with any gRNA comprising a plus strand targeting domain.

B. Targeted Integration via Homology-directed Repair (HDR)

[0260] In some of the embodiments provided herein, homology-directed repair (HDR) can be utilized for targeted integration of a specific portion of the template polynucleotide containing a transgene, e.g., nucleic acid sequence encoding a recombinant TCR, at a particular location in the genome, e.g., the TRAC locus. In some embodiments, the presence of a genetic disruption (e.g., a DNA break, such as described in Section I.A, including a second genetic disruption at a TRAC locus, described in Section I.A.2) and a template polynucleotide containing one or more homology arms (e.g., containing nucleic acid sequences homologous sequences surrounding the genetic disruption) can induce or direct HDR, with homologous sequences acting as a template for DNA repair. Based on homology between the endogenous gene sequence surrounding the genetic disruption and the 5’ and/or 3’ homology arms included in the template polynucleotide, cellular DNA repair machinery can use the template polynucleotide to repair the DNA break and resynthesize genetic information at the site of the genetic disruption, thereby effectively inserting or integrating the transgene sequences in the template polynucleotide at or near the site of the genetic disruption. In some embodiments, the genetic disruption, e.g., TRAC locus, can be generated by any of the methods for generating a targeted genetic disruption described herein.

[0261] Also provided are polynucleotides (in some aspects, referred to as “template polynucleotides”, e.g., comprising transgene sequences encoding a recombinant TCR), as described herein. In some embodiments, the provided polynucleotides can be employed in the methods described herein, e.g., involving HDR, to target transgene sequences encoding a recombinant TCR or a portion thereof, at the endogenous TRAC locus of the T cell.

[0262] In some embodiments, the template polynucleotide is or comprises a polynucleotide containing a transgene (exogenous or heterologous nucleic acids sequences) encoding a recombinant TCR or a portion thereof (e.g., one or more chain(s), region(s) or domain(s) of the recombinant TCR), and homology sequences (e.g., homology arms) that are homologous to sequences at or near the endogenous genomic site, e.g., at the endogenous TRAC locus. In some aspects, the template polynucleotide is introduced as a linear DNA fragment or comprised in a vector. In some aspects, the step for inducing genetic disruption and the step for targeted integration (e.g., by introduction of the template polynucleotide) are performed simultaneously or sequentially.

1. Homology-directed Repair (HDR)

[0263] In some embodiments, homology-directed repair (HDR) can be utilized for targeted integration or insertion of one or more nucleic acid sequences, e.g., transgene sequences, at one or more target site(s) in the genome, e.g., the TRAC locus. In some embodiments, the nuclease- induced HDR can be used to alter a target sequence, integrate a transgene at a particular target location, and/or to edit or repair a mutation in a particular target gene.

[0264] Alteration of nucleic acid sequences at the target site can occur by HDR with an exogenously provided polynucleotide (also referred to as donor polynucleotide or template sequence). For example, the template polynucleotide provides for alteration of the target sequence, such as insertion of the transgene contained within the template polynucleotide. In some embodiments, a plasmid or a vector can be used as a template for homologous recombination. In some embodiments, a linear DNA fragment can be used as a template for homologous recombination. In some embodiments, a single stranded template polynucleotide can be used as a template for alteration of the target sequence by alternate methods of homology directed repair (e.g., single strand annealing) between the target sequence and the template polynucleotide. Template polynucleotide-effected alteration of a target sequence depends on cleavage by a nuclease, e.g., a targeted nuclease such as CRISPR/Cas9. Cleavage by the nuclease can comprise a double strand break or two single strand breaks.

[0265] In some embodiments, “recombination” refers to a process of exchange of genetic information between two polynucleotides. In some embodiments, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a template polynucleotide to template repair of a target DNA (i.e., the one that experienced the double-strand break, e.g., target site in the endogenous gene), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the template polynucleotide to the target. In some embodiments, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the template polynucleotide, and/or “synthesis-dependent strand annealing,” in which the template polynucleotide is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the template polynucleotide is incorporated into the target polynucleotide. As described herein, the genetic disruption of the target site or target position can be created by any mechanisms, such as ZFNs, TALENs, CRISPR/Cas9 system, or TtAgo nucleases.

[0266] In some embodiments, double strand cleavage is effected by a nuclease, e.g., a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-terminal RuvC-like domain, e.g., a wild type Cas9.

[0267] In some embodiments, DNA repair mechanisms can be induced by a nuclease after (1) a single double-strand break, (2) two single strand breaks, (3) two double stranded breaks with a break occurring on each side of the target site, (4) one double stranded break and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target site (5) four single stranded breaks with a pair of single stranded breaks occurring on each side of the target site, or (6) one single stranded break. In some embodiments, a single-stranded template polynucleotide is used and the target site can be altered by alternative HDR.

[0268] Template polynucleotide-effected alteration of a target site depends on cleavage by a nuclease molecule. Cleavage by the nuclease can comprise a nick, a double strand break, or two single strand breaks, e.g., one on each strand of the DNA at the target site. After introduction of the breaks on the target site, resection occurs at the break ends resulting in single stranded overhanging DNA regions.

[0269] In canonical HDR, a double- stranded template polynucleotide is introduced, comprising homologous sequence to the target site that will either be directly incorporated into the target site or used as a template to insert the transgene or correct the sequence of the target site. After resection at the break, repair can progress by different pathways, e.g., by the double Holliday junction model (or double strand break repair, DSBR, pathway) or the synthesisdependent strand annealing (SDSA) pathway.

[0270] In some embodiments, other DNA repair pathways such as single strand annealing (SSA), single- stranded break repair (SSBR), mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), intrastrand cross-link (ICL), translesion synthesis (TLS), error-free postreplication repair (PRR) can be employed by the cell to repair a doublestranded or single-stranded break created by the nucleases.

2. Polynucleotide for Integration at a TRAC Locus

[0271] A polynucleotide, such as a template polynucleotide having homology with sequences at or near one or more target site(s) in the endogenous DNA can be used to alter the structure of a target DNA, e.g., targeted insertion of the transgene encoding a recombinant TCR or a portion thereof. In some embodiments, the template polynucleotide contains homology sequences (e.g., homology arms) flanking the transgene, e.g., nucleic acid sequences encoding a recombinant TCR or a portion thereof, for targeted insertion. In some embodiments, the homology sequences target the transgene at a TRAC locus. In some embodiments, the template polynucleotide includes additional sequences (coding or non-coding sequences) between the homology arms, such as a regulatory sequences, such as promoters and/or enhancers, splice donor and/or acceptor sites, internal ribosome entry site (IRES), sequences encoding ribosome skipping elements (e.g., 2A peptides), markers and/or SA sites, and/or one or more additional transgenes.

[0272] In some embodiments, the transgene contained in the polynucleotide, e.g., template polynucleotide, comprises a sequence encoding a recombinant TCR or a portion thereof. In some embodiments, the transgene can encode any of the recombinant TCRs described herein, for example, in Section III. A herein or any chains, regions and/or domains thereof. In some embodiments, the transgene encodes a recombinant T cell receptor (TCR) or any chains, regions and/or domains thereof. In some aspects, the transgene encodes a TCR alpha (TCRa) chain and a TCR beta (TCRP) chain of a recombinant TCR that binds to or recognizes a peptide epitope of human papillomavirus (HPV). In some aspects, the polynucleotide, e.g., template polynucleotide, comprises any transgene sequences provided herein or a nucleic acid sequence encoding any recombinant TCR described herein, e.g., in Section III.A.

[0273] In some embodiments, the polynucleotide, e.g., template polynucleotide, contains a transgene, encoding a recombinant TCR or chain thereof that contains one or more variable domains and one or more constant domains, for example, a variable alpha (Va) region and/or a variable beta (VP) region, and/or a constant alpha (Ca) region and/or a constant beta (CP) region of any of the recombinant TCRs described herein, e.g., in Section III.A.

[0274] In some aspects, the polynucleotide, e.g., template polynucleotide, comprises any transgene sequences provided herein or a nucleic acid sequence encoding any recombinant TCR described herein, e.g., in Section III.A. In some embodiments, the encoded recombinant TCR or chain thereof contains one or more constant domains that shares complete, e.g., at or about 100% identity, to all or a portion and/or fragment of an endogenous TCR constant domain. In some embodiments, the transgene encodes all or a portion of a constant domain, e.g., a portion or fragment of the constant domain that is completely or partially identical to an endogenous TCR constant domain.

[0275] In some embodiments, the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and/or a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:18.

[0276] In some embodiments, the transgene comprises a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18.

[0277] In some embodiments, the transgene contains nucleotides of a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion of the nucleic acid sequence set forth in any one of SEQ ID NO: 24 and 201- 204. In some embodiments, the transgene contains nucleotides of a sequence having at or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion of the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, the transgene comprises the nucleic acid sequence set forth in SEQ ID NO: 24.

[0278] In some of embodiments, the transgene contains a sequence encoding a TCRa and/or TCRP chain or a portion thereof that has been codon-optimized. In some embodiments, the transgene encodes a portion of a TCRa and/or TCRP chain with less than 100% amino acid sequence identity to a corresponding portion of a native or endogenous TCRa and/or TCRP chain. In some embodiments, the encoded TCRa and/or TCRP chain contains an amino acid sequence with, with about, or with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater than 99% identity but less than 100% identity to a corresponding native or endogenous TCRa and/or TCRP chain. In particular embodiments, the transgene encodes a TCRa and/or TCRP constant domain or portion thereof with less than 100% amino acid sequence identity to a corresponding native or endogenous TCRa and/or TCRP constant domain. In some embodiments, the TCRa and/or TCRP constant domain contains an amino acid sequence with, with about, or with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater than 99% identity but less than 100% identity to a corresponding native or endogenous TCRa and/or TCRP chain.

[0279] In some embodiments, the transgene contains one or more modifications(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

[0280] In some embodiments, the one or more non-native cysteine residues are capable of forming non-native disulfide bonds, e.g., with a TCRa chain encoded by the transgene. In some embodiments, one or more different template polynucleotides are used for targeting integration of the transgene at one or more different target sites. For targeting integration at different target sites, one or more genetic disruptions (e.g., DNA break) are generated at one or more of the target sites; and one or more different homology sequences are used for targeting integration of the transgene into the respective target site. In some embodiments, the transgene inserted at each site is the same or substantially the same. In some embodiments, transgene inserted at each site are different. In some embodiments, two or more different transgenes, encoding two or more different domains or chains of a protein, is inserted at one or more target sites.

[0281] In some embodiments, the transgene encodes all or a portion of a TCRa constant domain (Ca) and/or a TCRP constant domain (CP) with one or more modifications to remove or prevent a native disulfide bond, e.g., between the TCRP chain encoded by the transgene and the endogenous TCRa chain. In some embodiments, one or more native cysteines that form and/or are capable of forming a native interchain disulfide bond are substituted to another residue, e.g., serine or alanine. In some embodiments, the portion of a TCRa constant domain (Ca) and/or a TCRP constant domain (CP) is modified to replace one or more non-cysteine residues to a cysteine. Exemplary of modified encoded TCRa constant domain (Ca) and/or TCRP constant domain (CP) include any of those described herein, for example, in Section III.A, and those described in W02006/000830, WO 2006/037960 and Kuball et al. (2007) Blood, 109:2331- 2338.

[0282] In some embodiments, the transgene encoding the recombinant TCR or a portion thereof encodes one chain of a recombinant TCR and a second transgene encodes a different chain of the recombinant TCR. In some embodiments, the transgene encoding the recombinant TCR or a portion thereof encodes the alpha (TCRa) chain of the recombinant TCR and a second transgene encodes the beta (TCRP) chain of the recombinant TCR. In some embodiments, two or more transgene encoding different domains of the recombinant TCRs are targeted for integration at two or more target sites.

[0283] The sequence of interest in the template polynucleotide may comprise one or more sequences encoding a functional polypeptide (e.g., a cDNA), with or without a promoter.

[0284] In some embodiments, nuclease-induced HDR results in an insertion of a transgene (also called “exogenous sequence” or “transgene sequence”) for expression of a transgene for targeted insertion. The template polynucleotide sequence is typically not identical to the genomic sequence where it is placed. A template polynucleotide sequence can contain a non- homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, template polynucleotide sequence can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A template polynucleotide sequence can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a transgene and flanked by regions of homology to sequence in the region of interest.

[0285] In some aspects, nucleic acid sequences of interest, including coding and/or noncoding sequences and/or partial coding sequences, that are inserted or integrated at the target location in the genome can also be referred to as “transgene,” “transgene sequences,” “exogenous nucleic acids sequences,” “heterologous sequences” or “donor sequences.” In some aspects, the transgene is a nucleic acid sequence that is exogenous or heterologous to an endogenous genomic sequences, such as the endogenous genomic sequences at a specific target locus or target location in the genome, of a T cell, e.g., a human T cell. In some aspects, the transgene is a sequence that is modified or different compared to an endogenous genomic sequence at a target locus or target location of a T cell, e.g., a human T cell. In some aspects, the transgene is a nucleic acid sequence that originates from or is modified compared to nucleic acid sequences from different genes, species and/or origins. In some aspects, the transgene is a sequence that is derived from a sequence from a different locus, e.g., a different genomic region or a different gene, of the same species.

[0286] Polynucleotides for insertion can also be referred to as “transgene” or “exogenous sequences” or “donor” polynucleotides or molecules. The template polynucleotide can be DNA, single- stranded and/or double-stranded and can be introduced into a cell in linear or circular form. The template polynucleotide can be RNA single-stranded and/or double-stranded and can be introduced as a RNA molecule (e.g., part of an RNA virus). See also, U.S. Patent Publication Nos. 20100047805 and 20110207221. The template polynucleotide can also be introduced in DNA form, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the template polynucleotide can be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues are added to the 3’ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. If introduced in double- stranded form, the template polynucleotide may include one or more nuclease target site(s), for example, nuclease target sites flanking the transgene to be integrated into the cell’s genome. See, e.g., U.S. Patent Publication No. 20130326645.

[0287] In some embodiments, the double-stranded template polynucleotide includes sequences (also referred to as transgene) greater than 1 kb in length, for example between 2 and 200 kb, between 2 and 10 kb (or any value therebetween). The double-stranded template polynucleotide also includes at least one nuclease target site, for example. In some embodiments, the template polynucleotide includes at least 2 target sites, for example for a pair of ZFNs or TALENs. Typically, the nuclease target sites are outside the transgene sequences, for example, 5’ and/or 3’ to the transgene sequences, for cleavage of the transgene. The nuclease cleavage site(s) may be for any nuclease(s). In some embodiments, the nuclease target site(s) contained in the double- stranded template polynucleotide are for the same nuclease(s) used to cleave the endogenous target into which the cleaved template polynucleotide is integrated via homology-independent methods.

[0288] In some embodiments, the nucleic acid template system is double stranded. In some embodiments, the nucleic acid template system is single stranded. In some embodiments, the nucleic acid template system comprises a single stranded portion and a double stranded portion.

[0289] In certain embodiments, the polynucleotide, e.g., template polynucleotide contains and/or includes a transgene encoding all or a portion of a recombinant TCR, e.g., a chain of a recombinant TCR. In particular embodiments, the transgene is targeted at a target site(s) that is within a gene, locus, or open reading frame that encodes an endogenous receptor, e.g., an endogenous gene encoding one or more regions, chains or portions of a TCR.

[0290] In some embodiments, the template polynucleotide contains the transgene, e.g., recombinant TCR-encoding nucleic acid sequences, flanked by homology sequences (also called “homology arms”) on the 5’ and 3’ ends, to allow the DNA repair machinery, e.g., homologous recombination machinery, to use the template polynucleotide as a template for repair, effectively inserting the transgene into the target site of integration in the genome. The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the template polynucleotide. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In some embodiments, a homology arm does not extend into repeated elements, e.g., ALU repeats or LINE repeats.

[0291] Exemplary homology arm lengths include at least or at least about or is or is about 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500- 750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides. Exemplary homology arm lengths include less than or less than about or is or is about 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000- 2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

[0292] In some embodiments, the template polynucleotide comprises about 500 to 1000, e.g., 600 to 900 or 700 to 800, base pairs of homology on either side of the target site at the endogenous gene, such as a second target site at an endogenous TRAC locus. In some embodiments, the template polynucleotide comprises at least or less than or about 200, 300, 400, 500, 600, 700, 800, 900 or 1000 base pairs, homology 5’ of the target site, 3’ of the target site, or both 5’ and 3’ of the target site, e.g., within the TRAC gene, locus, or open reading frame (e.g., described in Table 3 herein). [0293] In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 base pairs homology 3’ of the target site. In some embodiments, the template polynucleotide comprises about 100 to 500, 200 to 400 or 250 to 350, base pairs homology 3’ of the transgene and/or target site. In some embodiments, the template polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 5’ of the target site, e.g., within the TRAC gene, locus, or open reading frame (e.g., described in Table 3 herein). In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 base pairs homology 5’ of the target site. In some embodiments, the template polynucleotide comprises about 100 to 500, 200 to 400 or 250 to 350, base pairs homology 5’ of the transgene and/or target site. In some embodiments, the template polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs homology 3’ of the target site, e.g., within the TRAC gene, locus, or open reading frame (e.g., described in Table 3 herein).

[0294] In some embodiments, a template polynucleotide is to a nucleic acid sequence which can be used in conjunction with one or more agent(s) capable of introducing a genetic disruption to alter the structure of a target site. In some embodiments, the target site is modified to have the some or all of the sequence of the template polynucleotide, typically at or near cleavage site(s). In some embodiments, the template polynucleotide is single stranded. In some embodiments, the template polynucleotide is double stranded. In some embodiments, the template polynucleotide is DNA, e.g., double stranded DNA In some embodiments, the template polynucleotide is single stranded DNA. In some embodiments, the template polynucleotide is encoded on the same vector backbone, e.g. AAV genome, plasmid DNA, as the Cas9 and gRNA. In some embodiments, the template polynucleotide is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In some embodiments, the template polynucleotide is on a separate polynucleotide molecule as the Cas9 and gRNA. In some embodiments, the Cas9 and the gRNA are introduced in the form of a ribonucleoprotein (RNP) complex, and the template polynucleotide is introduced as a polynucleotide molecule, e.g., in a vector. Types or nucleic acids and vectors for delivery include any of those described in Section I.C and Section II herein.

[0295] In some embodiments, the polynucleotide, e.g., template polynucleotide, alters the structure of the target site, e.g., insertion of transgene, by participating in a homology directed repair event. In some embodiments, the template polynucleotide alters the sequence of the target site. In some embodiments, the template polynucleotide includes sequence that corresponds to a site on the target sequence that is cleaved by one or more agent(s) capable of introducing a genetic disruption. In some embodiments, the template polynucleotide includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first agent capable of introducing a genetic disruption, and a second site on the target sequence that is cleaved in a second agent capable of introducing a genetic disruption.

[0296] In some embodiments, a template polynucleotide comprises the following components: [5’ homology arm] -[transgene] -[3’ homology arm]. The homology arms provide for recombination into the chromosome, thus insertion of the transgene into the DNA at or near the cleavage site, e.g., target site(s). In some embodiments, the homology arms flank the most distal target site(s).

[0297] In some embodiments, the 3’ end of the 5’ homology arm is the position next to the 5’ end of the transgene. In some embodiments, the 5’ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 5’ from the 5’ end of the transgene. In some embodiments, the 5’ end of the 3’ homology arm is the position next to the 3’ end of the transgene. In some embodiments, the 3’ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 3’ from the 3’ end of the transgene.

[0298] In some embodiments, for targeted insertion, the homology arms, e.g., the 5’ and 3’ homology arms, may each comprise about 1000 base pairs (bp) of sequence flanking the most distal gRNAs (e.g., 1000 bp of sequence on either side of the target site).

[0299] It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu repeats or LINE elements. For example, a 5’ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, a 3’ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5’ and the 3’ homology arms may be shortened to avoid including certain sequence repeat elements. It is contemplated herein that template polynucleotides for targeted insertion may be designed for use as a single-stranded oligonucleotide, e.g., a single-stranded oligodeoxynucleotide (ssODN). When using a ssODN, 5’ and 3’ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made. In some embodiments, a longer homology arm is made by a method other than chemical synthesis, e.g., by denaturing a long double stranded nucleic acid and purifying one of the strands, e.g., by affinity for a strand-specific sequence anchored to a solid substrate. [0300] Similarly, in some embodiments, the template polynucleotide has a 5’ homology arm, a transgene, and a 3’ homology arm, such that the template polynucleotide extends substantially the same distance on either side of the target site. For example, the homology arms may have different lengths, but the transgene may be selected to compensate for this. For example, the transgene may extend further 5’ from the target site than it does 3’ of the target site, but the homology arm 5’ of the target site is shorter than the homology arm 3’ of the target site, to compensate. The converse is also possible, e.g., that the transgene may extend further 3’ from the target site than it does 5’ of the target site, but the homology arm 3’ of the target site is shorter than the homology arm 5’ of the target site, to compensate.

[0301] The template polynucleotide can be linear single stranded DNA In some embodiments, the template polynucleotide is (i) linear single stranded DNA that can anneal to the nicked strand of the target DNA, (ii) linear single stranded DNA that can anneal to the intact strand of the target DNA, (iii) linear single stranded DNA that can anneal to the transcribed strand of the target DNA, (iv) linear single stranded DNA that can anneal to the non-transcribed strand of the target DNA, or more than one of the preceding.

[0302] In some embodiments, the template polynucleotide is a single stranded nucleic acid. In another embodiment, the template polynucleotide is a double stranded nucleic acid. In some embodiments, the template polynucleotide is linear double stranded DNA. The length may be, e.g., about 200-5000 nucleotides, e.g., about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. The length may be, e.g., at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, the length is no greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, a double stranded template polynucleotide has a length of about 160 nucleotides, e.g., about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800- 1800, 900-1700, 1000-1600, 1100-1500 or 1200-1400 nucleotides.

[0303] In some embodiments, the template polynucleotide is circular double stranded DNA, e.g., a plasmid. In some embodiments, the template polynucleotide comprises about 500 to 1000 nucleotides of homology on either side of the transgene and/or the target site. In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of homology 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene. In some embodiments, the template polynucleotide comprises at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of homology 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene. In some embodiments, the template polynucleotide comprises no more than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of homology 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene.

[0304] In some embodiments, the length of any of the polynucleotides, e.g., template polynucleotides, is at or about 200-10000 nucleotides, e.g., at or about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides, or a value between any of the foregoing. In some embodiments, the length is at least at or about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides, or a value between any of the foregoing. In some embodiments, the length is no greater than at or about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides. In some embodiments, the length is at or about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100-1500 or 1200-1400 nucleotides. In some embodiments, the polynucleotide is at least at or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length, or any value between any of the foregoing. In some embodiments, the polynucleotide is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length. In some embodiments, the polynucleotide is at or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length. The length is about 200-5000 base pairs, e.g., about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. The length is at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, the length is no greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, a single stranded template polynucleotide has a length of about 160 nucleotides, e.g., about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800- 1800, 900-1700, 1000-1600, 1100-1500 or 1200-1400 nucleotides.

[0305] In some embodiments, the template polynucleotide contains homology arms for targeting the endogenous TRAC locus (exemplary nucleotide sequence of the human TRAC gene locus set forth in SEQ ID NO: 278; NCBI Reference Sequence: NG_001332.3, TRAC or described in Table 3 herein). In some embodiments, the genetic disruption of the TRAC locus is introduced at early coding region the gene, including sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp), or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp). In some embodiments, the genetic disruption is introduced using any of the targeted nucleases and/or gRNAs described in Section I.A herein. In some embodiments, the template polynucleotide comprises about 500 to 1000, e.g., 600 to 900 or 700 to 800, nucleotides of homology on either side of the genetic disruption introduced by the targeted nucleases and/or gRNAs. In some embodiments, the template polynucleotide comprises about 500, 600, 700, 800, 900 or 1000 nucleotides of 5’ homology arm sequences, which is homologous to 500, 600, 700, 800, 900 or 1000 nucleotides of sequences 5’ of the genetic disruption (e.g., at TRAC locus), the transgene, and about 500, 600, 700, 800, 900 or 1000 nucleotides of 3’ homology arm sequences, which is homologous to 500, 600, 700, 800, 900 or 1000 nucleotides of sequences 3’ of the genetic disruption (e.g., at TRAC locus). In some embodiments, exemplary 5’ and 3’ homology arms for targeted integration at the TRAC locus are set forth in SEQ ID NO: 56 and 57, respectively. In some embodiments, exemplary 5’ and 3’ homology arms for targeted integration at the TRAC locus are set forth in SEQ ID NOS: 279-285 and 286-292, respectively.

[0306] In some instances, the template polynucleotide comprises a promoter, e.g., a promoter that is exogenous and/or not present at or near the target locus. In some embodiments in which the functional polypeptide encoding sequences are promoterless, expression of the integrated transgene is then ensured by transcription driven by an endogenous promoter or other control element in the region of interest.

[0307] The transgene, including the transgene encoding the recombinant TCR or a portion thereof, can be inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the transgene is inserted (e.g., TRAC). For example, the coding sequences in the transgene can be inserted without a promoter, but in-frame with the coding sequence of the endogenous target gene, such that expression of the integrated transgene is controlled by the transcription of the endogenous promoter at the integration site. In some embodiments, the transgene encoding the recombinant TCR or a portion thereof and/or the one or more second transgene independently is operably linked to the endogenous promoter of the gene at the target site. In some embodiments, a ribosome skipping element/self-cleavage element, such as a 2A element, is placed upstream of the transgene coding sequence, such that the ribosome skipping element/self-cleavage element is placed in-frame with the endogenous gene, such that the expression of the transgene encoding the recombinant or a portion thereof and/or the one or more second transgene is operably linked to the endogenous TCRa promoter.

[0308] In some embodiments, the transgene encoding the recombinant TCR or a portion thereof and/or the one or more second transgene independently comprises one or more multicistronic element(s). In some embodiments, the one or more multicistronic element(s) are upstream of the transgene encoding the recombinant TCR or a portion thereof and/or the one or more second transgene. In some embodiments, the multicistronic element(s) is positioned between the transgene encoding the recombinant TCR or a portion thereof and the one or more second transgene. In some embodiments, the multicistronic element(s) is positioned between the nucleic acid sequence encoding the TCRa or a portion thereof and the nucleic acid sequence encoding the TCRP or a portion thereof. In some embodiments, the ribosome skip element comprises a sequence encoding a ribosome skip element selected from among a T2A, a P2A, a E2A or a F2A or an internal ribosome entry site (IRES).

[0309] In some embodiments, the encoded TCRa chain and TCRP chain are separated by a linker region. In some embodiments, a linker sequence is included that links the TCRa and TCRP chains to form the single polypeptide strand. In some embodiments, the linker is of sufficient length to span the distance between the C terminus of the a chain and the N terminus of the P chain, or vice versa, while also ensuring that the linker length is not so long so that it blocks or reduces bonding to a target peptide-MHC complex. In some embodiments, the linker may be any linker capable of forming a single polypeptide strand, while retaining TCR binding specificity. In some embodiments, the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker between the TCRa chain or portion thereof and the TCRP chain or portion thereof that is recognized by and/or is capable of being cleaved by a protease. In certain embodiments, the linker between the TCRa chain or potion thereof and the TCRP chain or portion thereof contains a ribosome skipping element or a self-cleaving element.

[0310] In some embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRP chain]-[linker]-[TCRa chain]. In particular embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRP chain] - [self-cleaving element] -[TCRa chain]. In certain embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRP chain] -[ribosome skipping sequence]-[TCRa chain]. In some embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRa chain] -[linker] -[TCRP chain]. In particular embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRa chain]-[self-cleaving element] -[TCRP chain]. In certain embodiments, the transgene is or include a sequence of nucleotides that is or includes the structure [TCRa chain]- [ribosome skipping sequence] -[TCRP chain]. In some embodiments, the structures are encoded by a polynucleotide strand of a single or double stranded polynucleotide, in a 5’ to 3’ orientation.

[0311] In some cases, the ribosome skipping element/self-cleavage element, such as a T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C- terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). This allows the inserted transgene to be controlled by the transcription of the endogenous promoter at the integration site, e.g., TRAC promoter. Exemplary ribosome skipping element/self-cleavage element include 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 220), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 219), Thosea asigna virus (T2A, e.g., SEQ ID NO: 215 or 216), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 217 or 218) as described in U.S. Patent Publication No. 20070116690. In some embodiments, the template polynucleotide includes a P2A ribosome skipping element (sequence set forth in SEQ ID NO: 217 or 218) upstream of the transgene, e.g., recombinant TCR encoding nucleic acids or between the sequences encoding a TCRa chain and the sequences encoding a TCRP chain.

[0312] In some embodiments, transgene may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue- specific promoter. In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, any described herein, such as a human elongation factor la promoter (EFla). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is a tissue-specific promoter or a viral promoter. In some embodiments, the promoter is a non-viral promoter. In some embodiments, the promoter is a modified EFla promoter with HTLV1 enhancer, for example set forth in SEQ ID NO: 270. In some embodiments, the transgene does not include a regulatory element, e.g. promoter.

[0313] In some embodiments, a “tandem” cassette is integrated into the selected site. In some embodiments, one or more of the “tandem” cassettes encode one or more polypeptide or factors, each independently controlled by a regulatory element or all controlled as a multi- cistronic expression system. In some embodiments, such as those where the polynucleotide contains a first and second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operatively linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Patent No. 6,060,273). In some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three polypeptides separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin), as described herein. The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some embodiments, the “tandem cassette” includes the first component of the cassette comprising a promoterless sequence, followed by a transcription termination sequence, and a second sequence, encoding an autonomous expression cassette or a multi-cistronic expression sequence. In some embodiments, the tandem cassette encodes two or more different polypeptides or factors, e.g., two or more chains or domains of a recombinant TCR. In some embodiments, nucleic acid sequences encoding two or more chains or domains of the recombinant TCR are introduced as tandem expression cassettes or bi- or multi-cistronic cassettes, into one target DNA integration site.

[0314] The transgene may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. In some embodiments, the transgene (e.g., with or without peptide-encoding sequences) is integrated into any endogenous locus. In some embodiments, the transgene is integrated into an endogenous TRAC locus.

[0315] In some embodiments, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. Further, the control elements of the genes of interest can be operably linked to reporter genes to create chimeric genes (e.g., reporter expression cassettes). In an exemplary embodiment, the template polynucleotide includes homology arms for targeting at the TRAC locus, regulatory sequences, e.g., promoter, and nucleic acid sequences encoding a recombinant TCR.

[0316] In some embodiments, exemplary template polynucleotides contain transgene encoding a recombinant T cell receptor under the operable control of the human elongation factor 1 alpha (EFla) promoter with HTEV1 enhancer (sequence set forth in SEQ ID NO:270), 5’ homology arm sequence of approximately 600 bp (e.g., set forth in SEQ ID NO:56), 3’ homology arm sequence of approximately 600 bp (e.g., set forth in SEQ ID NO:57) that are homologous to sequences surrounding the target integration site in exon 1 of the human TCRa constant domain (TRAC) gene. In some embodiments, the template polynucleotide further contains other nucleic acid sequences, e.g., nucleic acid sequences encoding a marker, e.g., a surface marker or a selection marker. In some embodiments, the template polynucleotide further contains viral vector sequences, e.g., adeno-associated virus (AAV) vector sequences.

[0317] The transgene contained on the template polynucleotide described herein may be isolated from plasmids, cells or other sources using known standard techniques such as PCR. Template polynucleotide for use can include varying types of topology, including circular supercoiled, circular relaxed, linear and the like. Alternatively, they may be chemically synthesized using standard oligonucleotide synthesis techniques. In addition, template polynucleotides may be methylated or lack methylation. Template polynucleotides may be in the form of bacterial or yeast artificial chromosomes (BACs or YACs).

[0318] A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, template polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with materials such as a liposome, nanoparticle or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

[0319] In other aspects, the template polynucleotide is delivered by viral and/or non-viral gene transfer methods. In some embodiments, the template polynucleotide is delivered to the cell via an adeno associated virus (AAV), such as any described herein.

[0320] In some embodiments, the template polynucleotide is comprised in a viral vector, and is at least at or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length, or any value between any of the foregoing. In some embodiments, the polynucleotide is comprised in a viral vector, and is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length. In some embodiments, the polynucleotide is comprised in a viral vector, and is at or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length.

[0321] In some embodiments, the template polynucleotide is an adenovirus vector, e.g., an

AAV vector, e.g., a ssDNA molecule of a length and sequence that allows it to be packaged in an AAV capsid. The vector may be, e.g., less than 5 kb and may contain an ITR sequence that promotes packaging into the capsid. The vector may be integration-deficient. In some embodiments, the template polynucleotide comprises about 150 to 1000 nucleotides of homology on either side of the transgene and/or the target site. In some embodiments, the template polynucleotide comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene. In some embodiments, the template polynucleotide comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene. In some embodiments, the template polynucleotide comprises at most 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5’ of the target site or transgene, 3’ of the target site or transgene, or both 5’ and 3’ of the target site or transgene.

[0322] In some embodiments, the template polynucleotide is a lentiviral vector, e.g., an IDLV (integration deficiency lentivirus).

[0323] The double-stranded template polynucleotides described herein may include one or more non-natural bases and/or backbones. In particular, insertion of a template polynucleotide with methylated cytosines may be carried out using the methods described herein to achieve a state of transcriptional quiescence in a region of interest.

[0324] The polynucleotide may comprise any transgene of interest (exogenous sequence). Exemplary exogenous sequences include, but are not limited to any polypeptide coding sequence (e.g., cDNAs or fragments thereof), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and various types of expression constructs. Marker genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.

[0325] In some embodiments, the transgene comprises a polynucleotide encoding any polypeptide of which expression in the cell is desired, including, but not limited to antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the foregoing. In some embodiments, the coding sequences may be, for example, cDNAs.

[0326] In some embodiments, the transgene further encodes one or more marker(s). In some embodiments, the one or more marker(s) is a transduction marker, surrogate marker and/or a selection marker.

[0327] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells that have been introduced with the polynucleotide, e.g., a polynucleotide encoding a recombinant TCR. In some embodiments, the transduction marker can indicate or confirm modification of a cell. In some embodiments, the surrogate marker is a protein that is made to be co-expressed on the cell surface with the recombinant TCR. In particular embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant TCR. In some embodiments, the nucleic acid sequence encoding the recombinant TCR is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as a T2A, a P2A, an E2A or an F2A. Extrinsic marker genes may in some cases be utilized in connection with engineered cell to permit detection or selection of cells and, in some cases, also to promote cell suicide.

[0328] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

[0329] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0330] In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[0331] In an exemplary embodiment, the template polynucleotide is included as an adeno- associated virus (AAV) vector construct, containing a nucleic acid sequence encoding a recombinant TCRa and TCRP chains under the control of a constitutive promoter, flanked by homology arms of about 600 base pairs each on the 5’ and 3’ side of the nucleic acid sequence encoding the recombinant TCR for targeting at exon 1 of the endogenous TRAC gene. Exemplary 5’ homology arm for targeting at TRAC include the sequence set forth in SEQ ID NO: 56. Exemplary 3’ homology arm for targeting at TRAC include the sequence set forth in SEQ ID NO:57.

[0332] In some embodiments, the polynucleotide contains the structure: [5' homology arm]- [transgene sequence] -[3' homology arm]. In some embodiments, the polynucleotide contains the structure: [5' homology arm]-[multicistronic element] -[transgene sequence]-[3' homology arm]. In some embodiments, the polynucleotide contains the structure: [5' homology arm]- [promoter]- [transgene sequence] -[3' homology arm].

[0333] Construction of such expression cassettes, following the teachings of the present specification, utilizes methodologies well known in molecular biology (see, for example, Ausubel or Maniatis). Before use of the expression cassette to generate a transgenic animal, the responsiveness of the expression cassette to the stress-inducer associated with selected control elements can be tested by introducing the expression cassette into a suitable cell line (e.g., primary cells, transformed cells, or immortalized cell lines).

C. Delivery of Agents for Genetic Disruption and Template Polynucleotides

[0334] In some embodiments, the genetic disruption, such as a first genetic disruption at an endogenous TGFBR2 and/or a second genetic disruption at an endogenous TRAC locus is carried out by delivering or introducing one or more agent(s), such as a first agent and/or a second agent, capable of inducing a genetic disruption, e.g., Cas9 and/or gRNA components, to a cell, using any of a number of known delivery method or vehicle for introduction or transfer to cells, for example, using viral delivery vectors, or any of the known methods or vehicles for delivering Cas9 molecules and gRNAs. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497- 505. In some embodiments, nucleic acid sequences encoding one or more components of one or more agent(s) capable of inducing a genetic disruption is introduced into the cells, e.g., by any methods for introducing nucleic acids into a cell described herein or known. In some embodiments, a vector encoding components of one or more agent(s) capable of inducing a genetic disruption such as a CRISPR guide RNA and/or a Cas9 enzyme can be delivered into the cell.

[0335] In some embodiments, the one or more agent(s) capable of inducing a genetic disruption, e.g., one or more agent(s) that is a Cas9/gRNA, is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas9 protein or variant thereof. For example, the Cas9 protein is delivered as RNP complex that comprises a Cas9 protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, Calcium Phosphate transfection, cell compression or squeezing. In some embodiments, the RNP can cross the plasma membrane of a cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.). In some embodiments, delivery of the one or more agent(s) capable of inducing genetic disruption, e.g., CRISPR/Cas9, as an RNP offers an advantage that the targeted disruption occurs transiently, e.g., in cells to which the RNP is introduced, without propagation of the agent to cell progenies. For example, delivery by RNP minimizes the agent from being inherited to its progenies, thereby reducing the chance of off-target genetic disruption in the progenies. In such cases, the genetic disruption and the integration of transgene can be inherited by the progeny cells, but without the agent itself, which may further introduce off-target genetic disruptions, being passed on to the progeny cells.

[0336] Agent(s) and components capable of inducing a genetic disruption, e.g., a Cas9 molecule and gRNA molecule, can be introduced into target cells in a variety of forms using a variety of delivery methods and formulations, as set forth in Tables 5 and 6, or methods described in, e.g., WO 2015/161276; US 2015/0056705, US 2016/0272999, US 2017/0211075; or US 2017/0016027. As described further herein, the delivery methods and formulations can be used to deliver template polynucleotides and/or other agents to the cell (such as those required for engineering the cells) in prior or subsequent steps of the methods described herein. When a Cas9 or gRNA component is encoded as DNA for delivery, the DNA may typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression. Exemplary promoters for Cas9 molecule sequences include, e.g., CMV, EFla, EFS, MSCV, PGK, or CAG promoters. Useful promoters for gRNAs include, e.g., Hl, EF-la, tRNA or U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule may comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In some embodiments a promoter for a Cas9 molecule or a gRNA molecule may be, independently, inducible, tissue specific, or cell specific. In some embodiments, an agent capable of inducing a genetic disruption is introduced RNP complexes. Table 5. Exemplary Delivery Methods

Table 6. Comparison of Exemplary Delivery Methods

[0337] In some embodiments, DNA encoding Cas9 molecules and/or gRNA molecules, or RNP complexes comprising a Cas9 molecule and/or gRNA molecules, can be delivered into cells by known methods or as described herein. For example, Cas9-encoding and/or gRNA- encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof. In some embodiments, the polynucleotide containing the agent(s) and/or components thereof is delivered by a vector (e.g., viral vector/virus or plasmid). The vector may be any described herein.

[0338] In some aspects, a CRISPR enzyme (e.g. Cas9 nuclease) in combination with (and optionally complexed with) a guide sequence is delivered to the cell. For example, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. For example, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Staphylococcus aureus or Neisseria meningitides.

[0339] In some embodiments, a Cas9 nuclease (e.g., that encoded by mRNA from Staphylococcus aureus or from Streptococcus pyogenes, e.g. pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and a guide RNA specific to the target locus (e.g. TGFBR2 or TRAC locus in humans) are introduced into cells.

[0340] In some embodiments, the polynucleotide containing the agent(s) and/or components thereof or RNP complex is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA or RNA or proteins or combination thereof, e.g., ribonucleoprotein (RNP) complexes, can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Lett 12: 6322-27, Kollmannsperger et al (2016) Nat Comm 7, 10372), gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof. [0341] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas9-and/or gRNA-encoding DNA or RNP complex in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the Cas9-and/or gRNA-encoding DNA in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0342] In some embodiments, the delivery vehicle is a non- viral vector. In some embodiments, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., FesMnCh) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In some embodiments, the non-viral vector is an organic nanoparticle. Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG), and protamine-nucleic acid complexes coated with lipid. Exemplary lipids and polymers for gene transfer include those described in, for example, WO 2019/195492 and WO 2020/223535.

[0343] In some embodiments, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In some embodiments, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In some embodiments, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In some embodiments, a stimulus-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[0344] In some embodiments, the delivery vehicle is a biological non-viral delivery vehicle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coll), bacteria having nutritional and tissue- specific tropism to target specific cells, bacteria having modified surface proteins to alter target cell specificity). In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissuespecificity. In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes -subject-derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands).

[0345] In some embodiments, RNA encoding Cas9 molecules and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Let 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof.

[0346] In some embodiments, delivery via electroporation comprises mixing the cells with the RNA encoding Cas9 molecules and/or gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the RNA encoding Cas9 molecules and/or gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0347] In some embodiments, Cas9 molecules can be delivered into cells by known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (such as described in Lee, et al. (2012) Nano Let 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.

[0348] In some embodiments, the one or more agent(s) capable of introducing a cleavage, e.g., a Cas9/gRNA system, is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas9 protein or variant thereof. For example, the Cas9 protein is delivered as RNP complex that comprises a Cas9 protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, calcium phosphate transfection, cell compression or squeezing.

[0349] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas9 molecules with or without gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the Cas9 molecules with or without gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.

[0350] In some embodiments, delivery via electroporation comprises mixing the cells with the Cas9 molecules with or without gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, delivery via electroporation is performed using a system in which cells are mixed with the Cas9 molecules.

[0351] In some embodiments, the polynucleotide containing the agent(s) and/or components thereof is delivered by a combination of a vector and a non- vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer than either a viral or a liposomal method alone.

[0352] In some embodiments, more than one agent(s) or components thereof are delivered to the cell. For example, in some embodiments, agent(s) capable of inducing a genetic disruption of two or more locations in the genome, e.g., a first target site at a TGFBR2 locus and a second target site at a TRAC locus, are delivered to the cell. In some embodiments, agent(s) and components thereof are delivered using one method. For example, in some embodiments, one or more agents, for example, for inducing a first genetic disruption at a first target site at a TGFBR2 locus and a second genetic disruption at a second target site at a TRAC locus, are delivered as a first agent, e.g., a first RNP, and a second agent, e.g., a second RNP, respectively. In other embodiments, one or more agents, for example, for inducing a first genetic disruption at a first target site at a TGFBR2 locus and a second genetic disruption at a second target site at a TRAC locus, are delivered as polynucleotides encoding the components for genetic disruption. In some embodiments, one polynucleotide can encode agents that target a first target site at a TGFBR2 locus and a second target site at a TRAC locus. In some embodiments, two or more different polynucleotides can encode the agents that target a first target site at a TGFBR2 locus and a second target site at a TRAC locus. In some embodiments, the one or more agents, such as a first agent and a second agent, capable of inducing a genetic disruption can be delivered as ribonucleoprotein (RNP) complexes, and two or more different RNP complexes can be delivered together as a mixture, or separately. In some aspects, the two or more different RNP complexes, such as a first RNP targeting a first target site at a TGFBR2 locus and a second RNP targeting a second target site at a TRAC locus, are delivered together, such as electroporated together, for example, in one electroporation reaction.

[0353] In some embodiments, one or more polynucleotides other than the one or more agent(s) capable of inducing a genetic disruption and/or component thereof, e.g., one or more CRISPR-Cas combinations, such as a template polynucleotide for HDR-directed integration (such as any template polynucleotide described herein, e.g., in Section I.B.2), are delivered. In some embodiments, the polynucleotide, e.g., template polynucleotide, is delivered at the same time as one or more of the components of the Cas system. In some embodiments, the polynucleotide is delivered before or after (e.g., less than about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In some embodiments, the polynucleotide, e.g., template polynucleotide, is delivered by a different means from one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component. The polynucleotide, e.g., template polynucleotide, can be delivered by any of the delivery methods described herein. For example, the polynucleotide, e.g., template polynucleotide, can be delivered by a viral vector, e.g., any described herein such as an AAV vector, and the Cas9 component and/or the gRNA molecule component can be delivered by electroporation. In some embodiments, the polynucleotide, e.g., template polynucleotide, includes one or more exogenous sequences, e.g., transgene sequences that encode a recombinant TCR or a portion thereof and/or other exogenous gene nucleic acid sequences.

[0354] In some embodiments, the polynucleotide, e.g., a polynucleotide such as a template polynucleotide encoding the recombinant TCR, are introduced into the cells in nucleotide form, e.g., as a polynucleotide or a vector. In particular embodiments, the polynucleotide contains a transgene that encodes the recombinant TCR or a portion thereof.

[0355] In some embodiments, the polynucleotide, e.g., template polynucleotide, is introduced into the cell for engineering, in addition to the agent(s) capable of inducing a targeted genetic disruption, e.g., nuclease and/or gRNAs. In some embodiments, the polynucleotide(s) may be delivered prior to, simultaneously or after the agent(s) capable of inducing a targeted genetic disruption is introduced into a cell. In some embodiments, the polynucleotide(s) are delivered simultaneously with the agents. In some embodiments, the polynucleotides are delivered prior to the agents, for example, seconds to hours to days before the agents, including, but not limited to, 1 to 60 minutes (or any time therebetween) before the agents, 1 to 24 hours (or any time therebetween) before the agents or more than 24 hours before the agents. In some embodiments, the polynucleotides are delivered after the agents, seconds to hours to days after the agents, including immediately after delivery of the agent, e.g., between or between about between 30 seconds to 4 hours, such as about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after delivery of the agents and/or preferably within 4 hours of delivery of the agents. In some embodiments, the polynucleotide is delivered more than 4 hours after delivery of the agents. In some embodiments, the polynucleotides are delivered after the agents, for example, including, but not limited to, within 1 second to 60 minutes (or any time therebetween) after the agents, 1 to 4 hours (or any time therebetween) after the agents or more than 4 hours after the agents.

[0356] In some embodiments, the polynucleotides, e.g., template polynucleotides, may be delivered using the same delivery systems as the agent(s) capable of inducing a targeted genetic disruption, e.g., nuclease and/or gRNAs. In some embodiments, the polynucleotides may be delivered using different same delivery systems as the agent(s) capable of inducing a targeted genetic disruption, e.g., nuclease and/or gRNAs. In some embodiments, the polynucleotide is delivered simultaneously with the agent(s). In other embodiments, the polynucleotide is delivered at a different time, before or after delivery of the agent(s). Any of the delivery method described herein in Section I.C (e.g., in Tables 5 and 6) for delivery of nucleic acids in the agent(s) capable of inducing a targeted genetic disruption, e.g., nuclease and/or gRNAs, can be used to deliver the polynucleotide.

[0357] In some embodiments, the one or more agent(s) and the polynucleotide are delivered in the same format or method. For example, in some embodiments, the one or more agent(s) and the polynucleotide are both comprised in a vector, e.g., viral vector. In some embodiments, the polynucleotide is encoded on the same vector backbone, e.g. AAV genome, plasmid DNA, as the Cas9 and gRNA. In some aspects, the one or more agent(s) and the polynucleotide are in different formats, e.g., ribonucleic acid-protein complex (RNP) for the Cas9-gRNA agent and a linear DNA for the polynucleotide, but they are delivered using the same method. In some aspects, the one or more agent(s) and the polynucleotide are in different formats, e.g., ribonucleic acid-protein complex (RNP) for the Cas9-gRNA agent and the polynucleotide is in contained in an AAV vector, and the RNP is delivered using a physical delivery method (e.g., electroporation) and the polynucleotide is delivered via transduction of AAV viral preparations. In some aspects, the polynucleotide is delivered immediately after, e.g., within about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or 60 minutes after, the delivery of the one or more agent(s).

[0358] In some embodiments, the one or more agent(s) is or comprises a ribonucleoprotein (RNP) complex. In some embodiments, the concentration of the RNP incubated with, added to or contacted with the cells for engineering is at a concentration of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 pM, or a range defined by any two of the foregoing values. In some aspects, the concentration of the RNP is between at or about 1 pM and at or about 5 pM. In some embodiments, the concentration of the RNP incubated with, added to or contacted with the cells for engineering is at a concentration of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4 or 5 pM, or a range defined by any two of the foregoing values. In some aspects, the concentration of the RNP is between at or about 1.5 pM and at or about 2.5 pM. In some embodiments, the concentration of RNPs is at or about 2 pM.

[0359] In some aspects, the concentration of the RNP, such as the first RNP comprising a first gRNA targeting TGFBR2, and/or a second RNP comprising a second gRNA targeting TRAC, is between at or about 1 pM and at or about 5 pM, between at or about 1.5 pM and at or about 2.5 pM, between at or about 1.7 pM and at or about 2.5 pM, or between at or about 2 pM and at or about 2.5 pM. In some of any embodiments, the concentration of the RNP, such as the first RNP comprising a first gRNA targeting TGFBR2, and/or a second RNP comprising a second gRNA targeting TRAC is at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM, or a range defined by any two of the foregoing values. In some of any embodiments, the concentration of the RNP, such as a first gRNA targeting TGFBR2, is at or about 2.0 pM to at or about 2.5 pM. In some of any embodiments, the concentration of the RNP, such as a second RNP comprising a second gRNA targeting TRAC, is at or about 1.7 pM to at or about 2.5 pM. In some aspects, the concentration of the first RNP and/or the second RNP is between at or about 1 pM to at or about 5 pM. In some aspects, the concentration of the first RNP and/or the second RNP is at or about 1.5 pM. In some aspects, the concentration of the first RNP and/or the second RNP is at or about 1.7 pM. In some aspects, the concentration of the first RNP and/or the second RNP is at or about 2 pM. In some aspects, the concentration of the first RNP and/or the second RNP is at or about 2.2 pM. In some aspects, the concentration of the first RNP and/or the second RNP is at or about 2.5 pM.

[0360] In some embodiments, in the RNP complex, the ratio, e.g. the molar ratio, of the gRNA and the Cas9 molecule or other nucleases is at or about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, or a range defined by any two of the foregoing values. In some embodiments, in the RNP complex, the ratio, e.g., molar ratio, of the gRNA and the Cas9 molecule or other nucleases is at or about 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2:1 or 1:1, or a range defined by any two of the foregoing values.

[0361] In some embodiments, the polynucleotide is a linear or circular polynucleotide, such as a linear or circular DNA or linear RNA, and can be delivered using any of the methods described in Section I.C herein (e.g., Tables 5 and 6) for delivering polynucleotides into the cell.

[0362] In particular embodiments, the polynucleotide, e.g., the template polynucleotide, are introduced into the cells in nucleotide form, e.g., as or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, e.g., a DNA or RNA polynucleotide, that is suitable for transduction and/or transfection by any suitable and/or known non-viral method for gene delivery, such as but not limited to microinjection, electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof. In some embodiments, the non-viral polynucleotide is delivered into the cell by a non-viral method described herein, such as a non-viral method listed in Table 6 herein.

[0363] In some embodiments, the polynucleotide sequence can be comprised in a vector molecule containing sequences that are not homologous to the region of interest in the genomic DNA.

[0364] In some embodiments, the polynucleotides and sequences encoding the one or more agents may be on the same vector, for example an AAV vector. In some embodiments, the polynucleotides are delivered using an AAV vector and the one or more agents for inducing a genetic disruption, e.g., one or more CRISPR-Cas combination, are delivered as a different form, e.g., as mRNAs encoding the nucleases and/or gRNAs. In some embodiments, the polynucleotides and nucleases are delivered using the same type of method, e.g., a viral vector, but on separate vectors. In some embodiments, the polynucleotides are delivered in a different delivery system as the agents capable of inducing a genetic disruption, e.g., nucleases and/or gRNAs. In some embodiments, the polynucleotide is excised from a vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In some embodiments, the polynucleotide is on a separate polynucleotide molecule as the Cas9 and gRNA. In some embodiments, the Cas9 and the gRNA are introduced in the form of a ribonucleoprotein (RNP) complex, and the polynucleotide is introduced as a polynucleotide molecule, e.g., in a vector or a linear polynucleotide, e.g., linear DNA. Types or nucleic acids and vectors for delivery include any of those described in Section II herein.

II. NUCLEIC ACIDS AND VECTORS

[0365] In some embodiments, the one or more agent for genetic disruption and/or polynucleotides, e.g., template polynucleotides containing transgene encoding the recombinant TCR a portion thereof, are introduced into the cells in nucleic acid form, e.g., as polynucleotides and/or vectors. As described in Section I.C herein, the components for engineering can be delivered in various forms using various delivery methods, including as polynucleotides encoding the components. Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding one or more components of the one or more agent(s) capable of inducing a genetic disruption, and/or one or more template polynucleotides containing transgene, and vectors for genetically engineering cells for targeted integration of the transgene.

[0366] In some embodiments, provided are polynucleotides, e.g., template polynucleotides for targeting transgene at a specific genomic target location, e.g., at the TRAC locus. In some embodiments, provided are any template polynucleotides described in Section I.B.2 herein. In some embodiments, the template polynucleotide contains transgene that include nucleic acid sequences that encode a recombinant TCR or other polypeptides and/or factors, and homology arms for targeted integration. In some embodiments, the template polynucleotide can be contained in a vector.

[0367] In some embodiments, agents capable of inducing a genetic disruption can be encoded in one or more polynucleotides. In some embodiments, the component of the agents, e.g., CRISPR-Cas combination, can be encoded in one or more polynucleotides, and introduced into the cells. In some embodiments, the polynucleotide encoding one or more component of the agents can be included in a vector.

[0368] In some embodiments, a vector may comprise a sequence that encodes a CRISPR- Cas combination and/or template polynucleotides. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.

[0369] One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is or comprises a regulatable (e.g., inducible) promoter or a constitutive promoter, such as any described herein, for example, in Section I.B.2. In some embodiments, the polynucleotide and/or vector does not include a regulatory element, e.g. promoter. In some embodiments, the promoter is selected from among an RNA pol I, pol II or pol III promoter. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV, SV40 early region or adenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., a U6 or Hl promoter). In another embodiment, the promoter is a regulated promoter (e.g., inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog thereof or is capable of being bound by or recognized by a Lac repressor or a tetracycline repressor, or an analog thereof. In some embodiments, the promoter drives expression only in a specific cell type (e.g., a T cell or B cell or NK cell specific promoter).

[0370] Exemplary constitutive promoters include, e.g., simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EFla), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken P-Actin promoter coupled with CMV early enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (sequence set forth in SEQ ID NO:293 or 294; see Challita et al. (1995) J. Virol. 69(2):748-755) . In some embodiments, the promoter is a tissue- specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non- viral promoter. In some cases, the promoter is selected from among human elongation factor 1 alpha (EFla) promoter (sequence set forth in SEQ ID NO: 295 or 296) or a modified form thereof (EFla promoter with HTLV1 enhancer; sequence set forth in SEQ ID NO: 270) or the MND promoter (sequence set forth in SEQ ID NO: 293 or 294). In some aspects, the promoter is set forth in SEQ ID NO:270. [0371] In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In some embodiments, the virus is an RNA virus (e.g., ssRNA virus). Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno- associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses, or any of the viruses described elsewhere herein.

[0372] In some embodiments, the polynucleotide can be transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, the polynucleotide are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557) or HIV-1 derived lentiviral vectors.

[0373] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0374] In some embodiments, the virus infects dividing cells. In another embodiment, the virus infects non-dividing cells. In another embodiment, the virus infects both dividing and nondividing cells. In another embodiment, the virus can integrate into the host genome. In another embodiment, the virus is engineered to have reduced immunity, e.g., in human. In another embodiment, the virus is replication-competent. In another embodiment, the virus is replicationdefective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In another embodiment, the virus causes transient expression of the Cas9 molecule and/or the gRNA molecule for the purposes of transient induction of genetic disruption. In another embodiment, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

[0375] In some embodiments, the polynucleotide encoding the one or more agent(s) and/or containing the polynucleotide is delivered by a recombinant retrovirus. In another embodiment, the retrovirus (e.g., Moloney murine leukemia virus) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replication- competent. In another embodiment, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted.

[0376] In some embodiments, the polynucleotide encoding the one or more agent(s) and/or containing the polynucleotide is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication. In some embodiments, the lentivirus is an HIV-derived lentivirus.

[0377] In some embodiments, the polynucleotide encoding the one or more agent(s) and/or containing the polynucleotide is delivered by a recombinant adenovirus. In another embodiment, the adenovirus is engineered to have reduced immunity in humans.

[0378] In some embodiments, the polynucleotide encoding the one or more agent(s) and/or containing the polynucleotide is delivered by a recombinant AAV. In some embodiments, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In another embodiment, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods, include AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y731F and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV7, AAV8, AAV 8.2, AAV9, AAV.rhlO, modified AAV.rhlO, AAV.rh32/33, modified AAV.rh32/33, AAV.rh43, modified AAV.rh43, AAV.rh64Rl, modified AAV.rh64Rl, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods. Any AAV vector can be used, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and combinations thereof. In some instances, the AAV comprises LTRs that are of a heterologous serotype in comparison with the capsid serotype (e.g., AAV2 ITRs with AAV5, AAV6, or AAV8 capsids). The template polynucleotide may be delivered using the same gene transfer system as used to deliver the nuclease (including on the same vector) or may be delivered using a different delivery system that is used for the nuclease. In some embodiments, the template polynucleotide is delivered using a viral vector (e.g., AAV) and the nuclease(s) is(are) delivered in mRNA form. The cell may also be treated with one or more molecules that inhibit binding of the viral vector to a cell surface receptor as described herein prior to, simultaneously and/or after delivery of the viral vector (e.g., carrying the nuclease(s) and/or template polynucleotide).

[0379] In some embodiments, the polynucleotide encoding the one or more agent(s) and/or containing the polynucleotide is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein.

[0380] A packaging cell is used to form a virus particle that is capable of infecting a target cell. Such a cell includes a 293 cell, which can package adenovirus, and a \|/2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed, e.g., Cas9. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions are supplied in trans by the packaging cell line. Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0381] In some embodiments, the viral vector has the ability of cell type recognition. For example, the viral vector can be pseudotyped with a different/altemative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., genetic modification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibody, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).

[0382] In some embodiments, the viral vector achieves cell type specific expression. For example, a tissue- specific promoter can be constructed to restrict expression of the transgene (Cas9 and gRNA) in only a specific target cell. The specificity of the vector can also be mediated by microRNA-dependent control of transgene expression. In some embodiments, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutinin (HA) can be incorporated to increase viral uptake into cells. In some embodiments, the viral vector has the ability of nuclear localization. For example, a virus that requires the breakdown of the nuclear membrane (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells.

[0383] In some embodiments, the polynucleotide, e.g., template polynucleotide is a linear or circular polynucleotide, such as a linear or circular DNA or linear RNA, and can be delivered using any of the methods described in Section I.C herein (e.g., Tables 5 and 6) for delivering polynucleotides into the cell.

[0384] In some embodiments, the polynucleotide, e.g., the template polynucleotide, and/or nucleic acid sequences encoding one or more agents, are introduced into the cells in nucleotide form, e.g., as or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, e.g., a DNA or RNA polynucleotide, that is used for transduction and/or transfection by any known non-viral method for gene delivery, such as but not limited to microinjection, electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-penetrating peptides, or a combination thereof. In some embodiments, the non-viral polynucleotide is delivered into the cell by a non-viral method described herein, such as a non-viral method listed in Table 6 herein.

III. ENGINEERED CELLS EXPRESSING RECOMBINANT TCRS AND CELL COMPOSITIONS

[0385] In some of any of the provided embodiments, the engineered cells express a recombinant T cell receptor. In some aspects, the recombinant TCR can target or bind an epitope or region of a cancer antigen, such as a peptide epitope expressed on the surface of a cancer cell. In some aspects, the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV).

[0386] In some embodiments, the recombinant TCR expressed in the engineered cells comprises a TCR alpha (TCRa) chain and/or a TCR beta (TCRP) chain. In some aspects, the recombinant TCR comprises a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region.

A. Encoded T Cell Receptors (TCRs)

[0387] In some of any of the provided embodiments, the encoded recombinant TCR or antigen-binding fragments thereof recognize or bind an epitope or region of a cancer antigen, in the context of an MHC molecule. In some aspects, exemplary antigens for the recombinant TCR includes a peptide epitope expressed on the surface of a cancer cell and/or a cell infected with HPV or a cell that contains HPV DNA sequences. In some aspects, exemplary recombinant TCRs include those that bind to or recognize a peptide epitope of human papillomavirus (HPV), in the context of an MHC molecule. In some aspects, provided are recombinant TCRs or antigen binding fragment thereof or an antibody or antigen fragments thereof, and proteins such as chimeric molecules containing one or more of the foregoing, such as the chimeric receptors, e.g., TCR-like CARs.

[0388] In some embodiments, the recombinant TCR or antigen-binding fragment thereof binds to or recognizes an antigen expressed on the surface of the cell line designated SCC152 (ATCC® CRL-3240™), which is a cell line derived from a squamous cell carcinoma and that contains HPV DNA sequences. In some aspects, cytotoxic activity of T cells containing the recombinant TCRs, e.g., TCRs, is stimulated upon contact of such cells with target cells, expressing the antigen, such as cancer cells and/or those that express HPV 16, such as HPV 16 E7, e.g. SCC 152 cells. In some embodiments, among the provided TCRs or antigen-binding fragment thereof provided herein are those that bind or recognize a peptide epitope of HPV 16 in the context of an MHC (e.g. a peptide epitope of HPV 16 E7).

[0389] Among such recombinant TCRs or antigen-binding fragments thereof are TCRs or antigen-binding fragments thereof that contain any of the variable alpha (Va) regions and/or a variable beta (VP) regions as described, individually, or a sufficient antigen-binding portion of such chain(s). In some embodiment, the provided recombinant TCRs comprise a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region. In some embodiments, the provided recombinant TCR or antigen-binding fragment thereof (e.g. anti-HPV 16 E7 TCRs) contains a Va region sequence or sufficient antigen-binding portion thereof that contains a CDR-1, CDR-2 and/or CDR-3 as described. In some embodiments, the provided TCR or antigen-binding fragment thereof (e.g., anti-HPV 16 E7 TCRs) contains a VP region sequence or sufficient antigen-binding portion that contains a CDR-1, CDR-2 and/or CDR-3 as described. In some embodiments, the TCR or antigen-binding fragment thereof (e.g. anti-HPV 16 E7 TCRs) contains a Va region sequence that contains a CDR-1, CDR-2 and/or CDR-3 as described and contains a VP region sequence that contains a CDR-1, CDR-2 and/or CDR-3 as described. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.

[0390] HPV is a causative organism in most cases of cervical cancer and has been implicated in anal, vaginal, vulvar, penile, and oropharyngeal cancers, and other cancers. Generally, the HPV genome contains an early region containing six open reading frames (El, E2, E4, E5, E6 and E7), which encode proteins involved in cell transformation and replication, and a late region containing two open reading frames (LI and L2), which encode proteins of the viral capsid. In general, E6 and E7 are oncogenes that can affect cell cycle regulation and contribute to the formation of cancers. For instance, the E6 gene product can cause p53 degradation and the E7 gene product can cause retinoblastoma (Rb) inactivation.

[0391] In some aspects, the recombinant TCR binds to a peptide epitope derived from HPV 16 E7 protein and/or to a peptide epitope expressed on a cell infected with HPV. In some embodiments, the TCR is an anti-HPV- 16 TCR, such as an anti-HPV 16 E7 TCR. In some of any of the provided embodiments, exemplary anti-HPV TCR or an antigen-binding fragment thereof includes an anti-HPV TCR or an antigen-binding fragment thereof, or a domain, chain or region thereof, for example, a variable alpha (Va) region and/or a variable beta (VP) region thereof, described in WO 2019/195486, WO 2019/070541, WO 2018/067618, or WO 2015/184228.

[0392] In some aspects, the recombinant TCR recognizes or binds HPV 16 E7 epitopes in the context of an MHC molecule, such as an MHC Class I molecule. In some aspects, the MHC Class I molecule is a human leukocyte antigen (HLA)-A2 molecule, including any one or more subtypes thereof, e.g. HLA-A*0201, *0202, *0203, *0206, or *0207. In some cases, there can be differences in the frequency of subtypes between different populations. For example, in some embodiments, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency has been reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206 and 23% HLA-A*0203. In some embodiments, the MHC molecule is HLA-A*0201. [0393] In some embodiments, the TCR or antigen-binding fragment thereof recognizes or binds to an epitope or region of HPV 16 E7 or HPV 16 E6, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOS: 267 and 297-303, and as shown below in Table 7. Exemplary anti-HPV TCR that bind to a peptide epitope of HPV 16 E7 or HPV 16 E6 include those described in WO 2019/195486, WO 2019/070541, WO 2018/067618, or WO 2015/184228.

[0394] In some embodiments, the TCR or antigen-binding fragment thereof is isolated or purified or is recombinant. In particular embodiments, any of the provided TCR or antigenbinding fragment thereof is recombinant. In some aspects, the TCR or antigen-binding fragment thereof is human. In some embodiments, the recombinant TCR is monoclonal. In some aspects, the recombinant TCR is a single chain. In other embodiments, the recombinant TCR contains two chains. In some embodiments, the TCR or antigen-binding fragment thereof is expressed on the surface of a cell.

[0395] In some aspects, the provided recombinant TCRs have one or more specified functional features, such as binding properties, including binding to particular epitopes, and/or particular binding affinities as described.

[0396] In some embodiments, the recombinant, chimeric or engineered receptor expressed in the engineered T cell is a T cell receptor (TCR) or antigen-binding fragment thereof. In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a and P chains (also known as TCRa and TCRp, respectively) or y and 6 chains (also known as TCRy and TCRS, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to an antigen, e.g., a peptide antigen or peptide epitope bound to an MHC molecule. In some embodiments, the TCR is in the aP form. Typically, TCRs that exist in aP and y6 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form.

Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens, such as peptides bound to major histocompatibility complex (MHC) molecules.

[0397] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing the a chain and P chain. In some embodiments, the TCR is an antigen-binding portion that is less than a full- length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a (Va) chain and variable P (VP) chain of a TCR, or antigen-binding fragments thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0398] In some embodiments, the variable domains of the TCR contain complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity of the peptide, MHC and/or MHC-peptide complex. In some embodiments, a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., lores et al., Proc. Nat'l Acad. Sci. U.S.A. 57:9138, 1990; Chothia et al., EMBO J. 737^5, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides. In some contexts, CDR1 of the beta chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the P-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426). [0399] In some embodiments, the TCRa chain and/or TCRP chain also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 ^rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain (e.g. alpha or beta) of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR, for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g. CD3y, CD36, CD3s and CD3(^ chains) contain one or more immunoreceptor tyrosine-based activation motif or IT AM and generally are involved in the signaling capacity of the TCR complex.

[0400] The various domains or regions of a TCR can be identified. In some cases, the exact locus of a domain or region can vary depending on the particular structural or homology modeling or other features used to describe a particular domain. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO: used to describe domain organization of a TCR are for illustrative purposes and are not meant to limit the scope of the embodiments provided. In some cases, the specific domain (e.g. variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some aspects, residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see e.g. www.imgt.org; see also, Lefranc et al. (2003) Developmental and Comparative Immunology, 27(l);55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 27-38, inclusive, the CDR2 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 56-65, inclusive, and the CDR3 sequences within a TCR Va region and/or VP region correspond to the amino acids present between residue numbers 105-117, inclusive.

[0401] Provided herein are anti-HPV 16 E7 (11-19) TCRs or antigen-binding fragments thereof. In some cases, the TCR recognizes or binds a peptide epitope derived from HPV 16 E7 that is or contains E7(l l-19) YMLDLQPET (SEQ ID NO: 267). In some embodiments, the

TCR recognizes or binds HPV 16 E7(l 1-19) in the context of an MHC, such as an MHC class I, e.g., HLA-A2. In some embodiments, the provided TCRs or antigen-binding fragments thereof are capable of or bind to a HPV 16 E7 (11-19)- peptide-MHC tetramer complex. In some aspects, engineered T cells containing or expressing such a TCR or antigen-binding fragment thereof exhibits cytotoxic activity upon contact with a cancer target cell and/or a target cell infected with HPV or that contains HPV DNA sequences, e.g. SCC152 cell.

[0402] In some embodiments, the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:10 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the Va region comprises a CDR-1 comprising the sequence of SEQ ID NO: 10.

[0403] In some embodiments, the Va region comprises a complementarity determining region 1 (CDR-2) comprising the sequence of SEQ ID NO: 11 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the Va region comprises a CDR-2 comprising the sequence of SEQ ID NO: 11.

[0404] In some embodiments, the Va region comprises a complementarity determining region 1 (CDR-3) comprising the sequence of SEQ ID NO: 12 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the Va region comprises a CDR-3 comprising the sequence of SEQ ID NO: 12.

[0405] In some embodiments, the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12.

[0406] In some instances, the Va region contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences contained within a Va region sequence set forth in SEQ ID NO:8.

[0407] In some instances, the Va region comprises the sequence set forth in SEQ ID NO:8, or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the Va region comprises the sequence set forth in SEQ ID NO:8.

[0408] In some embodiments, the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:3 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the VP region comprises a CDR-1 comprising the sequence of SEQ ID NO:3.

[0409] In some embodiments, the VP region comprises a complementarity determining region 1 (CDR-2) comprising the sequence of SEQ ID NO:4 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the VP region comprises a CDR-2 comprising the sequence of SEQ ID NO:4.

[0410] In some embodiments, the VP region comprises a complementarity determining region 1 (CDR-3) comprising the sequence of SEQ ID NO:5 or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the VP region comprises a CDR-3 comprising the sequence of SEQ ID NO:5.

[0411] In some embodiments, the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR-3 comprising the sequence of SEQ ID NO:5.

[0412] In some instances, the VP region contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences contained within a Va region sequence set forth in SEQ ID NO:1.

[0413] In some instances, the VP region comprises the sequence set forth in SEQ ID NO:1, or a sequence that comprises at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto. In some embodiments, the VP region comprises the sequence set forth in SEQ ID NO: 1.

[0414] In some embodiments, the Va region comprises a CDR-1 comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region comprises a CDR-1 comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR-3 comprising the sequence of SEQ ID NO:5.

[0415] In some embodiments, the TCR or antigen-binding fragment includes a Va region that contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences set forth in Table 8 and a VP region that contains a CDR- 1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, the CDR-2, and the CDR-3 amino acid sequences set forth in Table 8. Exemplary TCRs containing such CDRs, or their modified versions as described elsewhere herein, also are set forth in the Table 8. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.

[0416] In some embodiments, the Va region comprise the sequence of SEQ ID NO:8, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0417] In some embodiments, the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

[0418] In some embodiments, the TCRa chain and/or TCRP chain each further contain a constant domain. In some embodiments, the a chain constant domain (Ca) and P chain constant domain (CP) individually are mammalian, such as is a human or murine constant domain. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs.

[0419] In some aspects, provided herein are TCRs that contains a human constant region, such as an alpha chain containing a human Ca region and a beta chain containing a human Cp. In some embodiments, the provided TCRs are fully human. Among the provided TCRs are TCRs containing a human constant region, such as fully human TCRs, whose expression and/or activity, such as when expressed in human cells, e.g. human T cells, such as primary human T cells, are not impacted by or are not substantially impacted by the presence of an endogenous human TCR. In particular, it is observed herein that certain TCRs, such as the exemplary TCR designated TCR1; or a TCR containing Va set forth in SEQ ID NO:8 and the VP set forth in SEQ ID NO: 1; or a TCR containing the Va region comprises a CDR-1 comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region comprises a CDR-1 comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR- 3 comprising the sequence of SEQ ID NO:5, when formatted with a human constant region exhibit substantial activity in primary human T cells containing an endogenous TCR. In some embodiments, such TCRs containing a human constant region are not out-competed by the endogenous human TCR, such as for components of the CD3 complex.

[0420] In some embodiments, the TCR may be a heterodimer of two chains TCRa and TCRP that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, the constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, a TCR may have an additional cysteine residue in each of the TCRa and TCRP chains, such that the TCR contains two disulfide bonds in the constant domains. In some embodiments, each of the constant and variable domains contains disulfide bonds formed by cysteine residues.

[0421] In some embodiments, the TCR can contain an introduced disulfide bond or bonds. In some embodiments, the native disulfide bonds are not present. In some embodiments, the one or more of the native cysteines (e.g. in the constant domain of the a chain and P chain) that form a native interchain disulfide bond are substituted to another residue, such as to a serine or alanine. In some embodiments, an introduced disulfide bond can be formed by mutating noncysteine residues on the alpha and beta chains, such as in the constant domain of the a chain and P chain, to cysteine. Opposing cysteines in the TCRa and TCRP chains provide a disulfide bond that links the constant regions of TCRa and TCRP chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted constant region in which the native disulfide bonds are present, such as unsubstituted native human constant region or the unsubstituted native mouse constant region. In some embodiments, the presence of non-native cysteine residues (e.g. resulting in one or more non-native disulfide bonds) in a recombinant TCR can favor production of the desired recombinant TCR in a cell in which it is introduced over expression of a mismatched TCR pair containing a native TCR chain. In some embodiments, the TCRa and/or TCRP chain and/or a TCRa and/or TCRP chain constant domains are modified to replace one or more non-cysteine residues to a cysteine.

[0422] In some embodiments, the one or more non-native cysteine residues are capable of forming non-native disulfide bonds, e.g., between the recombinant TCRa and TCRP chain encoded by the transgene. In some embodiments, the cysteine is introduced at one or more of residue Thr48, Thr45, TyrlO, Thr45, and Serl5 with reference to numbering of a TCRa constant domain (Ca) set forth in SEQ ID NO: 168. In certain embodiments, cysteines can be introduced at residue Ser57, Ser77, Serl7, Asp59, of Glul5 of the TCRP chain with reference to numbering of TCRP constant domain (CP) set forth in SEQ ID NO: 173. Exemplary non-native disulfide bonds of a TCR are described in W02006/000830, WO 2006/037960 and Kuball et al. (2007) Blood, 109:2331-2338. In some embodiments, the transgene encodes a portion of a TCRa chain and/or a TCRa constant domain containing one or more modifications to introduce one or more disulfide bonds. In some embodiments, cysteines can be introduced or substituted at a residue corresponding to Thr48 of the Ca chain and Ser57 of the CP chain, at residue Thr45 of the Ca chain and Ser77 of the CP chain, at residue TyrlO of the Ca chain and Serl7 of the CP chain, at residue Thr45 of the Ca chain and Asp59 of the CP chain and/or at residue Serl5 of the Ca chain and Glul5 of the CP chain with reference to numbering of a Ca set forth in SEQ ID NO: 168, or a CP set forth in SEQ ID NO: 173. [0423] In some embodiments, the TCRa constant domain (Ca) of the recombinant TCR encoded by the transgene contains a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 168. In some embodiments, the TCRa constant domain has an amino acid sequence set forth in any of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence of amino acids that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto containing one or more cysteine residues capable of forming a non-native disulfide bond with a TCRP chain.

[0424] In some embodiments, the TCRP constant domain (CP) of the recombinant TCR encoded by the transgene contains a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 173. In some embodiments, the TCRP constant domain has an amino acid sequence set forth in any of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183, or a sequence of amino acids that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto containing one or more cysteine residues capable of forming a non-native disulfide bond with a TCRa chain.

[0425] In some embodiments, the Ca region comprises a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181 and/or the CP region comprises a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183. In some embodiments, the Ca region comprises a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178- 181; and the CP region comprises a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183.

[0426] In some embodiments, the Ca region comprises the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2. In some embodiments, the Ca region comprises the sequence of SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2.

[0427] In some embodiments, such TCRs containing a human constant region are expressed at similar or improved levels on the cell surface, exhibit the similar or greater functional activity (e.g. cytolytic activity) and/or exhibit similar or greater anti-tumor activity, when expressed by human cells that contain or express an endogenous human TCR, such as human T cells, as compared to the level of expression, functional activity and/or anti-tumor activity of a similar TCR containing the same VP and Va regions but that is formatted with a mouse constant region when expressed in the human cells. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, is expressed on the cell surface at a level that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the level of expression of a similar TCR containing the same VP and Va regions but that is formatted with a mouse constant region when expressed in the human T cells. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits an antigen-dependent functional activity, such as cytolytic activity, that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the activity of a similar TCR containing the same VP and Va regions but that is formatted with a mouse constant region when expressed in the human T cells. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits anti-tumor activity, such as when administered in vivo to a subject, that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the anti-tumor activity of a similar TCR containing the same VP and Va regions but that is formatted with a mouse constant region when expressed in the human T cells.

[0428] Exemplary TCRs or antigen-binding fragments include those set forth in Table 9, such as in each row therein. In some embodiments, the Va and VP regions contain the amino acid sequences corresponding to the SEQ ID NOs: set forth in Table 9, such as in each row therein. In some embodiments, the Va and VP regions contain the CDR-1, the CDR-2 and the CDR-3 sequences contained within the Va and VP regions set forth in Table 9, such as in each row therein. In some aspects, the TCR contains constant alpha and constant beta region sequences, such as those corresponding to the SEQ ID NOs: set forth in Table 9, such as in each row therein. In some cases, the TCR contains a full sequence comprising the variable and constant chain, such as a sequence corresponding to the SEQ ID NOs: set forth in Table 9 (“Full”), such as in each row therein. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such sequences, or their modified versions as described elsewhere herein, also are set forth in the Table 9, respectively, such as in each row therein.

[0429] Among the TCRs provided herein is a recombinant TCR containing a Va having a CDR1, 2 and 3 set forth in SEQ ID NOS: 10, 11, and 12, respectively, and a VP having a CDR1, 2, and 3 set forth in SEQ ID NOS: 3, 4, and 5, respectively. In some embodiments, the recombinant TCR contains a Va set forth in SEQ ID NO: 8 and a VP set forth in SEQ ID NO: 1. In some embodiments, the recombinant TCR contains a Ca region and a CP region. In some embodiments, the Ca and CP are human constant regions or are functional variants thereof. In some embodiments, the recombinant TCR contains an alpha chain containing a Ca human constant region or a variant thereof containing a non-native cysteine replacement, such as any described herein. In some embodiments, the recombinant TCR contains a Ca constant region set forth in SEQ ID NO:9. In some embodiments, the recombinant TCR contains a CP human constant region or a variant thereof containing a non-native cysteine replacement, such as any described herein. In some embodiments, the recombinant TCR contains a beta chain containing a CP region set forth in SEQ ID NO:2. In some embodiments, such as TCR contains a Ca set forth in SEQ ID NO:9 and a CP set forth in SEQ ID NO:2. In some embodiments, the recombinant TCR contains an alpha chain set forth in SEQ ID NO: 14 and a beta chain set forth in SEQ ID NO:7. In some embodiments, the recombinant TCR contains a TCRa chain set forth in SEQ ID NO: 14 and a TCRP chain set forth in SEQ ID NO:7. In some embodiments, the TCR is encoded by a polynucleotide that encodes the sequence set forth in SEQ ID NO:23. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any of such sequences.

[0430] In some embodiments, the variant comprises the amino acid sequence of any of the TCRs described herein with one, two, three, or four or more amino acid substitution(s) in the constant region of the alpha or beta chain. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the respective sequences of the region, domain or chain described herein. In some embodiments, the TCRs (or functional portions thereof) comprising the substituted amino acid sequence(s) advantageously provide one or more of decreased mis-pairing with an endogenous TCR chain(s), increased expression by a host cell, increased recognition of HPV 16 targets, and increased anti-tumor activity as compared to the parent TCR comprising an unsubstituted amino acid sequence. [0431] In some embodiments, such TCRs containing a human constant region are expressed at similar or improved levels on the cell surface, exhibit the similar or greater functional activity (e.g. cytolytic activity) and/or exhibit similar or greater anti-tumor activity, when expressed by human cells that contain or express an endogenous human TCR, such as human T cells, as compared to the level of expression, function activity and/or anti-tumor activity of the same TCR in similar human cells but in which expression of the endogenous TCR has been reduced or eliminated. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, is expressed on the cell surface at a level that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the level of expression of the same TCR when expressed in similar human T cells but in which expression of the endogenous TCR has been reduced or eliminated. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits an antigendependent functional activity, such as cytolytic activity, that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the activity as the same TCR expressed in similar human cells but in which expression of the endogenous TCR has been reduced or eliminated. In some examples a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits anti-tumor activity, such as when administered in vivo to a subject, that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the anti-tumor activity of the same TCR when expressed in similar human cells but in which expression of the endogenous TCR has been reduced or eliminated. In some of any of the above embodiments, a cell in which expression of the endogenous TCR has been reduced or eliminated can include cells in which the genes and/or gene products encoding the TCR, such as TRAC, is reduced, deleted, eliminated, knocked-out or disrupted.

B. Cells and Preparation of Cells for Genetic Engineering

[0432] In some embodiments, provided are engineered cells, e.g., genetically engineered or modified cells, and methods of engineering cells, including genetically engineered cells comprising a genetic disruption at a target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and a modified TRAC locus that comprises a transgene sequence encoding a recombinant TCR or a portion thereof. In some embodiments, polynucleotides, e.g., template polynucleotides, such as any of the template polynucleotides described herein, such as in Section I.B.2, containing nucleic acid sequences encoding a recombinant TCR or a portion thereof and/or additional molecule(s), are introduced into one a cell for engineering, e.g., according to the methods of engineering described herein. [0433] In some aspects, the transgene sequences (exogenous or heterologous nucleic acid sequences) in the polynucleotides and/or portions thereof are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acid sequences are not naturally occurring, such as a nucleic acid sequences not found in nature or is modified from a nucleic acid sequence found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0434] In some aspects, provided are method of producing a genetically engineered T cell, the method involving introducing any of the provided polynucleotides, e.g., described herein in Section I.B.2, into a T cell comprising a genetic disruption at a TGFBR2 locus. In some embodiments, upon performance of the methods, the expression of the endogenous TGFBR2 is reduced or eliminated, or a non-functional and/or partial sequence of TGFBR2 is expressed.

[0435] In some aspects, the genetic disruption is introduced by any agents or methods for introducing a targeted genetic disruption, including any described herein, such as in Section I. A.

[0436] In some aspects, the method produces a modified TRAC locus, said modified TRAC locus comprising a nucleic acid sequence encoding the recombinant TCR. In some aspects, provided are method of producing a genetically engineered T cell that involves introducing, into a T cell, one or more agent(s) capable of inducing a genetic disruption at a first target site within an endogenous TGFBR2 locus and/or a TRAC locus of the T cell; and introducing any of the provided polynucleotides, e.g., described herein in Section I.B.2, into a T cell comprising a genetic disruption at a TGFBR2 locus and/or a TRAC locus, wherein the method produces a modified TRAC locus, said modified TRAC locus comprising a nucleic acid sequence encoding the recombinant TCR, such as a recombinant TCR. In some embodiments, the nucleic acid sequence comprises a transgene sequence encoding the recombinant TCR or a portion thereof, such as any described herein, for example, in Section I.B.2. In some embodiments, the nucleic acid sequence comprises a transgene sequence encoding the recombinant TCR or a portion thereof, and the transgene sequence is targeted for integration within the endogenous TRAC locus via homology directed repair (HDR).

[0437] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as iPSCs. In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0438] Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as THI cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0439] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0440] In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the nucleic acid encoding a recombinant receptor such as a recombinant TCR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

[0441] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0442] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0443] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.

[0444] In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

[0445] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0446] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0447] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0448] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0449] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0450] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0451] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0452] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and/or CD45RO ⁺ T cells, are isolated by positive or negative selection techniques.

[0453] For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using anti-CD3/anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[0454] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker ⁺ ) at a relatively higher level (marker ¹¹¹⁸¹¹ ) on the positively or negatively selected cells, respectively.

[0455] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4 ⁺ or CD8 ⁺ selection step is used to separate CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In some aspects, the separated CD4 ⁺ and CD8 ⁺ populations or further separated populations are combined at a specific ratio, such as CD4+ T cells to CD8+ T cells ratio of from at or about 1:3 to at or about 3:1, such as at or about 1:1, prior to further engineering the cells to introduce the one or more genetic disruptions and/or to introduce the polynucleotides comprising transgenes encoding the recombinant receptor.

[0456] In some embodiments, CD8 ⁺ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8 ⁺ T cells and CD4 ⁺ T cells further enhances efficacy.

[0457] In embodiments, memory T cells are present in both CD62L ⁺ and CD62L’ subsets of CD8 ⁺ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0458] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8 ⁺ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8 ⁺ cell population or subpopulation, also is used to generate the CD4 ⁺ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0459] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4 ⁺ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD 14 and CD45RA or CD 19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order. [0460] CD4 ⁺ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4 ⁺ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 ⁺ T lymphocytes are CD45RO’, CD45RA ⁺ , CD62L ⁺ , CD4 ⁺ T cells. In some embodiments, central memory CD4 ⁺ cells are CD62L ⁺ and CD45RO ⁺ . In some embodiments, effector CD4 ⁺ cells are CD62L’ and CD45RO’.

[0461] In one example, to enrich for CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0462] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0463] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0464] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0465] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0466] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0467] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0468] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0469] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Pat. App. Pub. No. W02009/072003 or US 20110003380.

[0470] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0471] In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[0472] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[0473] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0474] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5) :355— 376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0475] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0476] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0477] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0478] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0479] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti- CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulating agents include IL-2, IL- 15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

[0480] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9): 651— 660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689- 701.

[0481] In some embodiments, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0482] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0483] In embodiments, antigen-specific T cells, such as antigen- specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

[0484] Various methods for the introduction of genetically engineered components, e.g., agents for inducing a genetic disruption and/or nucleic acids encoding recombinant receptors, e.g., recombinant TCRs, are known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the polypeptides or receptors, including via viral vectors, e.g., retroviral or lentiviral, non-viral vectors or transposons, e.g. Sleeping Beauty transposon system. Methods of gene transfer can include transduction, electroporation or other method that results into gene transfer into the cell, or any delivery methods described in Section EC or Section II herein. Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in WO2014055668 and U.S. Patent No. 7,446,190.

[0485] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (such as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0486] In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.

[0487] In some contexts, it may be desired to safeguard against the potential that overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) could potentially result in an unwanted outcome or lower efficacy in a subject, such as a factor associated with toxicity in a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11 :223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0488] In some embodiments, the cells, e.g., T cells, may be engineered either during or after expansion. This engineering for the introduction of the gene of the desired polypeptide or receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the CD3/CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus (e.g. via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in form of a peptide/MHC molecule, the cognate (cross-linking) ligand of the recombinant (e.g. natural ligand of a TCR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant domains within the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0489] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US 94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14-17.

[0490] As described herein, in some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, propagation and/or freezing for preservation, e.g. cryopreservation.

C. Composition of Cells Expressing a Recombinant TCR

[0491] Also provided are plurality or populations of the engineered cells, compositions containing such cells and/or enriched for such cells. In some aspects, the provided engineered cells and/or composition of engineered cells include any described herein, e.g., comprising a genetic disruption at a TGFBR2 locus and/or a modified TRAC locus comprising a transgene sequence encoding a recombinant TCR or a portion thereof, and/or are produced by the methods described herein. In some aspects, the plurality or population of engineered cells contain any of the engineered cells described herein, e.g., in Section III.C herein. In some aspects, the provided cells and cell composition can be engineered using any of the methods described herein, e.g., using agent(s) or methods for introducing genetic disruption, for example, as described in Section I.A herein, and/or using polynucleotides, such as template polynucleotide descried herein, for example in Section I.B.2, via homology-directed repair (HDR). In some aspects, such cell population and/or compositions provided herein is or are comprised in a pharmaceutical composition or a composition for therapeutic uses or methods, for example, as described in Section IV and Section V herein.

[0492] In some embodiments, the provided compositions comprises cells in which cells comprising a genetic disruption at the TGFBR2 locus make up at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition, or cells of a certain type, such as T cells or CD8+ or CD4+ cells. In some aspects, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells do not encode a functional TGFBR2 polypeptide, does not encode a functional TGFBR2 polypeptide, do not encode a TGFBR2 polypeptide, do not encode a full length TGFBR2 polypeptide, or the expression of TGFBR2 polypeptide is reduced or eliminated in the cell, and/or TGFP signal transmission is reduced or eliminated in the cell. In some embodiments, at least 80%, or more of the total cells in the composition, or cells of a certain type, such as T cells or CD8+ or CD4+ cells, comprise a genetic disruption at the TGFBR2 locus.

[0493] In some embodiments, the provided compositions containing cells in which cells expressing the recombinant TCR make up at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition, or cells of a certain type, such as T cells or CD8+ or CD4+ cells. In some embodiments, at least 75% or more of the total cells in the composition, or cells of a certain type, such as T cells or CD8+ or CD4+ cells, express the recombinant TCR.

[0494] In some embodiments, at least or greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the cells in the composition comprise a genetic disruption at a target site within a gene encoding a domain or region of T cell receptor alpha constant (TRAC) gene and/or does not express a gene product of an endogenous TRAC locus. In some embodiments, at least or greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the cells in the composition does not express a gene product of an endogenous TRAC locus. In some embodiments, at least or greater than 95% of the cells in the composition does not express a gene product of an endogenous TRAC locus.

[0495] In some embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus. In some embodiments, at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR. In some embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells do not express a gene product of an endogenous TRAC locus.

[0496] In some embodiments, provided are cell population and/or compositions that include a plurality of engineered immune cells expressing a recombinant TCR, wherein the nucleic acid sequence encoding the recombinant TCR is present at the TRAC locus, e.g., by integration of a transgene encoding recombinant TCR or a portion thereof at the TRAC locus via homology directed repair (HDR). In some embodiments, at least or greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells in the composition and/or cells in the composition that contains a genetic disruption at the TGFBR2 locus comprise integration of the transgene encoding recombinant TCR or a portion thereof at the TRAC locus. In some embodiments, the provided compositions containing cells such as in which cells expressing the recombinant TCR make up at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition that contains a genetic disruption at the TGFBR2 locus.

[0497] In some of any embodiments, the first genetic disruption is a genetic disruption of the TGFBR2 locus that is a knockout of the TGFBR2 gene, the second genetic disruption is a genetic disruption of the TRAC locus that is a knockout of the TRAC gene, and the recombinant TCR is knocked in to the TRAC locus by homology directed repair. In some of any embodiments, at least at or about 80% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by any of the provided methods do not express a gene product of an endogenous TRAC locus.

[0498] In some of any embodiments, among a population of cells containing a plurality of engineered T cells generated by any of the provided methods greater than 85% are knocked out for TRAC (TRAC KO), greater than 80% are knocked out for TGFBR2 (TGFBR2 KO) and greater than 65% have knock-in of the recombinant TCR. In some of any embodiments, among a population of cells containing a plurality of engineered T cells generated by any of the provided methods greater than 90% are knocked out for TRAC (TRAC KO), greater than 85% are knocked out for TGFBR2 (TGFBR2 KO) and greater than 70% have knock-in of the recombinant TCR. In some of any embodiments, among a population of cells containing a plurality of engineered T cells generated by any of the provided methods greater than 95% are knocked out for TRAC (TRAC KO), greater than 90% are knocked out for TGFBR2 (TGFBR2 KO) and greater than 75% have knock-in of the recombinant TCR.

[0499] In some of any embodiments, knock-in of the recombinant TCR is determined by a PCR-based method, such as ddPCR. In some of any embodiments, knock-in of the recombinant TCR is determined by flow cytometry for expression of the recombinant TCR. In some of any embodiments, knock-out of the endogenous TGFBR2 locus or TRAC locus is determined molecularly, such as by PCR-based methods (e.g. ddPCR) or next genome sequencing (NGS).

[0500] In some embodiments, at least at or about 70% of the engineered T cells in a composition or a plurality of T cells comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 50% of the engineered T cells in a composition or a plurality of T cells express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 70% of the engineered T cells in a composition or a plurality of T cells do not express a gene product of an endogenous TRAC locus.

[0501] In some embodiments, at least at or about 80% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells, or of the total cells or total T cells, in a composition containing a plurality of engineered T cells do not express a gene product of an endogenous TRAC locus.

[0502] In some embodiments, at least at or about 80% of the total cells or total T cells, in a composition containing a plurality of engineered T cells comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the total cells or total T cells, in a composition containing a plurality of engineered T cells express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the total cells or total T cells, in a composition containing a plurality of engineered T cells do not express a gene product of an endogenous TRAC locus.

[0503] In some embodiments, the provided cell population and/or compositions containing engineered cells include a cell population that exhibits more improved, uniform, homogeneous and/or stable expression and/or antigen binding by the recombinant TCR, e.g., exhibit reduced coefficient of variation, compared to the expression and/or antigen binding of cell populations and/or compositions generated using other methods. In some embodiments, the cell population and/or compositions exhibit at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower coefficient of variation of expression of the recombinant TCR and/or antigen binding by the recombinant TCR compared to a respective population generated using other methods, e.g., random integration of sequences encoding the recombinant TCR. The coefficient of variation is defined as standard deviation of expression of the nucleic acid of interest (e.g., transgene sequences encoding a recombinant TCR or a portion thereof) within a population of cells, for example CD4+ and/or CD8+ T cells, divided by the mean of expression of the respective nucleic acid of interest in the respective population of cells. In some embodiments, the cell population and/or compositions exhibit a coefficient of variation that is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35 or 0.30 or less, when measured among CD4+ and/or CD8+ T cell populations that have been engineered using the methods provided herein.

[0504] In some embodiments, the provided cell population and/or compositions containing engineered cells include a cell population that exhibits minimal or reduced random integration of the transgene encoding a recombinant TCR or a portion thereof. In some aspects, random integration of transgene into the genome of the cell can result in adverse effects or cell death due to integration of the transgene into undesired location in the genome, e.g., into an essential gene or a gene critical in regulating the activity of the cell, and/or unregulated or uncontrolled expression of the receptor. In some aspects, random integration of the transgene is reduced by at least or greater than 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to cell populations generated using other methods.

[0505] In some of any provided embodiments, a population of cells containing a plurality of engineered T cells contains greater than 75% T cells, such as greater than 80% T cells, greater than 85% T cells, greater than 90% T cells or greater than 95% T cells. In some embodiments, the T cells are CD3+. In some embodiments, the T cells include CD4+ and CD8+ T cells. In some embodiments, the ratio of CD4+ and CD8+ T cells in the population, or among cells expressing the recombinant TCR, is between at about 1:5 and 5:1, such as 1:3 and 3:1 or 1:2 and 2:1, such as at or about 1:1.

[0506] In some aspects, the composition of cells comprises CD4+ T cells and/or CD8+ T cells. In some aspects, the composition of cells comprises CD4+ T cells and CD8+ T cells. In some aspects, the percentage of CD4+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition. In some aspects, the percentage of CD8+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition. In some embodiments, the percentage of CD4+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition; and the percentage of CD8+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition. In some aspects, the composition comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1. In some aspects, the composition comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is at or about 1:1.

D. Assessment of Engineered T Cells and Compositions

[0507] In some of the embodiments, the methods include assessing the T cells or T cell compositions engineered to express the recombinant TCRs for particular properties. For example, the methods include assessing the T cells or T cell compositions for cell surface expression of the recombinant TCR and/or for recognition of a peptide in the context of an MHC molecule. For example, in any of the embodiments provided herein, functional assays can be performed on the T cells or T cell compositions expressing the recombinant TCR, generated or produced using any of the methods provided herein. In some embodiments, assays to detect functionality of the TCRs and activity of TCR signaling can also be performed.

[0508] In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, are less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), when administered to a subject having a disease or disorder. In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, are less sensitive to or resistant to immune suppression mediated by TGFp, when administered to a subject having a disease or disorder. In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder. In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, result in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder. In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, exhibit a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR. In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, result in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder, In some aspects, the provided engineered T cells or pharmaceutical compositions comprising the engineered T cells, result in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

[0509] In some embodiments, the T cells or T cell compositions are assessed for cell surface expression of the recombinant TCR, e.g., for the ability or capability to express a functional TCR, such as TCRaP, on the surface of the cell. In some embodiments, the T cells or T cell compositions are assessed for the ability or capability of the expressed TCRs for recognition of a peptide in the context of an MHC molecule, e.g., binding antigens or epitopes in the context of an MHC molecule. In some embodiments, the methods include assessing the T cells or T cell compositions for T cell activity and/or functionality. In some embodiments, the T cells or T cell compositions are assessed for is expression of the marker for transduction or introduction of the transgene.

[0510] In some embodiments, the T cells or T cell compositions are assessed for cell surface expression of the recombinant TCR, e.g., for the ability or capability to express a functional TCR, such as TCRaP, on the surface of the cell. In some embodiments, assessing surface expression of the TCR comprises contacting cells of each T cell composition with a binding reagent specific for the TCRa chain or the TCRP chain and assessing binding of the reagent to the cells. In some embodiments, the binding reagent is an antibody. In some embodiments, the binding reagent is detectably labeled, optionally fluorescently labeled, directly or indirectly. In some embodiments, the binding reagent is a fluorescently labeled antibody, such as an antibody labeled directly or indirectly. In some embodiments, the binding reagent is an anti-pan-TCR VP antibody or is an anti-pan-TCR Va antibody. In some embodiments, the binding reagent recognizes a specific family of chains. In some embodiments, the binding reagent is an anti- TCR VP or anti-TCR Va antibody that recognizes or binds a specific family, such as an anti- TCR VP22 antibody or an anti-TCR VP2 antibody. In some embodiments, the expression is detected using antibodies against one or more common portions, e.g., extracellular portions, of the TCR. For example, expression of TCR on the surface of the cell can be detected using pan- reactive anti-TCR antibodies, such as a pan-reactive TCR VP antibody, or a pan-reactive TCR Va antibody. Pan-reactive antibodies can detect the TCR regions regardless of its antigen or epitope binding specificity. In some embodiments, the cells are stained using a binding reagent, e.g., a labeled antibody that recognizes TCR cell surface expression, such as a fluorescently labeled pan-reactive TCR Va antibody or antigen -binding fragment thereof, and detecting using fluorescence microscopy, flow cytometry or fluorescence activated cell sorting (FACS). In some embodiments, T cells or T cell compositions that express the TCR on the surface of the cell, e.g., stain positive using pan-reactive anti-TCR antibodies, such as a pan-reactive TCR VP antibody, or a pan-reactive TCR Va antibody, are identified and/or selected.

[0511] In some embodiments, the T cells or T cell compositions are assessed for the ability or capability of the expressed TCRs for recognition of a peptide in the context of an MHC molecule, e.g., binding antigens or epitopes in the context of an MHC molecule. For example, in some embodiments, assessing the T cells or T cell compositions for recognition of a peptide in the context of an MHC molecule comprises: (1) contacting the cells or the cells of the T cell composition with a target antigen comprising a peptide-MHC complex and (2) determining the presence or absence of binding of the peptide-MHC complex to the cells and/or determining the presence or absence of T cell activation of the TCR-expressing cells upon engagement with the peptide-MHC complex.

[0512] In some embodiments, the T cells or T cell compositions to which nucleic acid sequences encoding recombinant TCRs are introduced, are tested by confirming that the recombinant TCRs bind to the desired or known antigen, such as a TCR ligand (MHC-peptide complex). In some embodiments, the binding of the cells to an antigen or an epitope can be detected by a number of methods. In some methods, a particular antigen, e.g. MHC-peptide complex, can be detectably labeled so that binding to the receptor, e.g. TCR, can be visualized. In some embodiments, the antigen can be soluble or expressed in a soluble form. In some embodiments, the TCR ligand can be a peptide-MHC tetramer, and in some cases the peptide- MHC tetramer can be detectably labeled, such as labeled with a fluorescent label. The peptide- MHC tetramer can be labeled directly or indirectly. In some embodiments, the fluorescent label can be detected using flow cytometry or fluorescence activated cell sorting (FACS) or fluorescence microscopy. In some embodiments, the methods include identifying one or more T cells or T cell compositions that recognize the peptide in the context of the MHC molecule, i.e. peptide-MHC complex.

[0513] In some cases, the binding of TCR, such as a recombinant TCR, to a peptide epitope, e.g. in complex with an MHC, results in or effects a functional property of the interaction. For example, a T cell expressing a TCR, such as a recombinant TCR, when specifically bound to an MHC-peptide complex, can induces a signal transduction pathway in the cell, induce cellular expression or secretion of an effector molecule (e.g. cytokine), reporter or other detectable readout of the interaction, or induce T cell activation or a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response. In some embodiments, the TCR, such as a recombinant TCR, can specifically bind to and immunologically recognize a peptide epitope, such that binding to the peptide epitope elicits an immune response.

[0514] Methods of testing a TCR for the ability to recognize a peptide epitope of a target polypeptide and for antigen specificity are known. In some embodiments, T cells or T cell compositions produced in accord with the provided method are contacted with a peptide-MHC complex, either in soluble form or via co-culture with peptide pulsed antigen presenting cells (e.g. T2 cells or other known antigen presenting cell that matches the MHC allele of the recombinant TCR). Exemplary antigens and MHC alleles of recombinant TCRs are described in Section III.A. In some embodiments, the methods include assessment of properties such as functional properties, of the recombinant TCR. In some embodiments, the method includes assessing T cell activation via the recombinant TCR, for example, determining the presence or absence of T cell activation of the TCR-expressing cells upon engagement with the peptide- MHC complex. In some embodiments, a readout of T cell activation by such methods includes release of cytokines (e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM- CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IE-2)). In addition, TCR function can be evaluated by measurement of cellular cytotoxicity, as described in Zhao et al., J. Immunol., 174:4415-4423 (2005).

[0515] In some embodiments, assessing T cell activation includes assessing activity or expression of a nucleic acid molecule encoding a reporter, e.g. a T cell activation reporter, assessing release of cytokines, and/or assessing functional activity of the T cell.

[0516] In some embodiments, the one or more assays involve one or more instrumentation, type of result or analysis, and/or read-outs. In some embodiments, the one or more assays are performed using fluorescently labeled reagents, such as antibodies directly or indirectly labeled with fluorophores, and are detected using a flow cytometry or fluorescence activated cell sorting (FACS) instrument. For example, for flow cytometry or FACS, multiple different fluorophores that have different peak excitation and emission wavelength can be detected. Thus, multiple fluorophore labels can be used to assess multiple properties, for example, expression of the TCR, recognition of the peptide in the context of an MHC molecule and/or T cell activation reporter expression, in one experimental reaction. In some embodiments, the one or more assays are performed in a high-throughput, multiplexed and/or large-scale manner.

[0517] In some embodiments, the methods further include assessing aspects of T cell activation, such as assessing release of cytokines and/or assessing functional activity of the T cell, e.g., cytolytic activity and/or helper T cell activity. In some embodiments, the assessments can be performed in T cells or T cell compositions generated using the embodiments described herein.

[0518] In some embodiments, the functional assays ca be performed in primary T cells, such as those isolated directly from a subject and/or isolated from a subject and frozen, such as primary CD4+ and/or CD8+ T cells, that have been engineered employing the embodiments provided herein.

[0519] In some embodiments, the methods include performing functional assays or detecting function of the TCR or the T cell. For example, functional assays for determining TCR activity or T cell activity include detection of cytokine secretion, cytolytic activity and/or helper T cell activity. For example, assessment of T cell activation includes assessing release of cytokines, and/or assessing functional activity of the T cell. In some embodiments, upon binding of the TCR to an antigen or an epitope, the cytoplasmic domain or intracellular signaling domain of the TCR activates at least one of the normal effector functions or responses of an immune cell, e.g., T cell engineered to express the TCR. For example, in some contexts, the TCR induces a function of a T cell such as cytolytic activity and/or helper T cell activity, such as secretion of cytokines or other factors. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.

[0520] In some embodiments, T cells or T cell compositions containing the recombinant TCRs are assessed for an immunological readout, such as using a T cell assay. In some embodiments, the TCR-expressing cells can activate a CD8+ T cell response. In some embodiments, CD8+ T cell responses can be assessed by monitoring CTL reactivity using assays that include, but are not limited to, target cell lysis via tumor spheroid assay, ⁵¹ Cr release, target cell lysis assays using real-time imaging reagents, target cell lysis assays using apoptosis detection reagent (e.g., Caspase 3/7 reagent), or detection of interferon gamma release, such as by enzyme-linked immunosorbent spot assay (ELISA), intracellular cytokine staining or ELISPOT. In some embodiments, the TCR-expressing cells can activate a CD4+ T cell response. In some aspects, CD4+ T cell responses can be assessed by assays that measure proliferation, such as by incorporation of [ ³ H] -thymidine into cellular DNA and/or by the production of cytokines, such as by ELISA, intracellular cytokine staining or ELISPOT. In some cases, the cytokine can include, for example, interleukin-2 (IL-2), interferon-gamma (IFN- gamma), interleukin-4 (IL-4), TNF-a, interleukin-6 (IL-6), interleukin- 10 (IL- 10), interleukin- 12 (IL- 12) or TGF p. In some embodiments, recognition or binding of the peptide epitope, such as a MHC class I or class II epitope, by the TCR can elicit or activate a CD8+ T cell response and/or a CD4+ T cell response.

[0521] In some embodiments, T cells or T cell compositions containing the recombinant TCRs are assessed for activity by administration in an in vivo animal model systems, e.g., a tumor xenograft model system.

IV. METHODS OF ADMINISTRATION AND USES IN THERAPY

[0522] Also provided are methods of administering and uses, such as therapeutic and prophylactic uses, of the recombinant TCRs, including TCRs and antigen-binding fragments thereof and antibodies or antigen-binding fragments thereof, and/or engineered cells expressing the recombinant TCRs. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with HPV, e.g., HPV 16, and/or in which cells or tissues express, e.g., specifically express, HPV 16, e.g., HPV 16 E7. In some embodiments, the molecule, cell, and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the recombinant TCRs and cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the recombinant TCRs or cells, or compositions comprising the same, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0523] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0524] As used herein, “delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0525] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.

[0526] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, a recombinant TCR or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the recombinant TCR or composition or cell.

[0527] An “effective amount” of an agent, e.g., a pharmaceutical formulation, recombinant TCR, or cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0528] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, recombinant TCR, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the recombinant TCRs, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.

[0529] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. [0530] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

[0531] Among the diseases to be treated are cancers. In some embodiments, the cancer is an HPV-associated cancers, and any HPV-associated, e.g., HPV 16-associated, diseases or conditions or diseases or conditions in which an HPV oncoprotein, e.g., E7, such as an HPV 16 oncoprotein, e.g., HPV 16 E7 is expressed. In certain diseases and conditions, the viral protein such as the oncoprotein such as the HPV 16 E7 is expressed in or by malignant cells and cancers, and/or a peptide epitope thereof is expressed on such malignant cancers or tissues, such as by way of MHC presentation. In some embodiments, the disease or condition is an HPV 16- expressing cancer. In some embodiments, the cancer is a carcinoma, melanoma or other precancerous or cancerous state caused by or otherwise associated with HPV, such as HPV- 16. In some embodiments, the carcinoma can be a squamous cell or adenocarionma. In some embodiments, the disease or condition can be characterized by an epithelial cell abnormality associated with oncogenic HPV infection, such as koilocytosis; hyperkeratosis; precancerous conditions encompassing intraepithelial neoplasias or intraepithelial lesion; high-grade dysplasias; and invasive or malignant cancers. Among the HPV 16-associated diseases or conditions that can be treated include, but are not limited to, cervical cancer, uterine cancer, anal cancer, colorectal cancer, vaginal cancer, vulvar cancer, penile cancer, oropharyngeal cancers, tonsil cancer, pharyngeal cancers (pharynx cancer), laryngeal cancer (larynx cancer), oral cancer, skin cancer, esophageal cancer, head and neck cancer such as a squamous cell carcinoma (SCC) head and neck cancer, or small cell lung cancer. In some embodiments, the disease or condition is a cervical cancer. In some embodiments, the disease or condition is a cervical carcinoma.

[0532] In some embodiments, the methods may include steps or features to identify a subject who has, is suspected to have, or is at risk for developing an HPV 16-associated disease or disorder (see e.g. U.S. Patent Nos. 6,355,424 and 8,968,995) and/or the subject to be treated may be a subject identified to have or to be so at risk for having or developing such HPV-associated disease or condition or cancer. Hence, provided in some aspects are methods for identifying subjects with diseases or disorders associated with HPV 16 E7 expression and selecting them for treatment and/or treating such subjects, e.g., selectively treating such subjects, with a provided HPV 16 recombinant TCR, including in some aspects with cells engineered to express such recombinant TCRs, including in some aspects any of the HPV 16 E7 TCRs or antigen binding fragments thereof or E7 antibodies, e.g., antibody fragments and proteins containing the same, such as the chimeric receptors, e.g., TCR-like CARs, and/or engineered cells expressing the TCRs or CARs.

[0533] For example, a subject may be screened for the presence of a disease or disorder associated with HPV 16 E7 expression, such as an HPV 16 E7-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of an HPV 16 E7- associated disease, e.g. a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with HPV 16 E7 expression and assayed for the expression level of HPV 16 E7. In some aspects, a subject who tests positive for an HPV 16 E7-associated disease or disorder may be selected for treatment by the present methods, and may be administered a therapeutically effective amount of cells expressing the recombinant TCR, or a pharmaceutical composition thereof as described herein. In some embodiments, the methods can be used to monitor the size or density of an HPV 16 E7-expressing tissue, e.g. tumor, over time, e.g., before, during, or after treatment by the methods. In some aspects, subjects treated by methods provided herein have been selected or tested positive for HPV expression according to such methods, e.g., prior to initiation of or during treatment.

[0534] In some embodiments, administration of a HPV 16 recombinant TCR, including any of the HPV 16 E7 TCRs or antigen binding fragments thereof or cells expressing the same can be combined with another therapeutic for the treatment of an HPV disease. For example, the additional therapeutic treatment can include treatment with another anti-cancer agent for the treatment of cervical cancer. Suitable dosages for such a co-administered agent may be lowered due to the combined action (synergy) of the agent and the provide HPV 16 recombinant TCR.

[0535] In some aspects, additional therapeutic agent is administered together with the recombinant TCR, engineered cells or cell compositions, such as pharmaceutical compositions, comprising the engineered cells. In some aspects, the additional therapeutic agent is IL-2. In some aspects, the additional therapeutic agent is aldesleukin (Proleukin).

[0536] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another HPV 16-specific recombinant TCR and/or cells expressing an HPV 16- targeting recombinant TCR and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another HPV 16-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. [0537] In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing TCRs, including the provided recombinant TCRs, the degree of immunogenicity in some embodiments is reduced compared to TCRs, including a different recombinant TCR.

[0538] In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided recombinant TCRs are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in an HPV 16-targeted manner, such that the cells of the disease or disorder are targeted for destruction.

[0539] Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in an HPV 16 E7- expressing cancer.

[0540] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0541] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0542] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0543] In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).

[0544] The provided recombinant TCRs or antigen-binding fragments thereof, and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.

[0545] For the prevention or treatment of disease, the appropriate dosage of the recombinant TCR or cell may depend on the type of disease to be treated, the type of recombinant TCR, the severity and course of the disease, whether the recombinant TCR is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the recombinant TCR, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

[0546] In certain embodiments, in the context of genetically engineered cells expressing the recombinant TCRs, a subject is administered the range of at or about one million to at or about 200 billion cells, such as, e.g., 1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), or such as at or about 10 million to at or about 100 billion cells (e.g., at or about 20 million cells, at or about 30 million cells, at or about 40 million cells, at or about 60 million cells, at or about 70 million cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases at or about 100 million cells to at or about 50 billion cells (e.g., at or about 120 million cells, at or about 250 million cells, at or about 350 million cells, at or about 450 million cells, at or about 650 million cells, at or about 800 million cells, at or about 900 million cells, at or about 3 billion cells, at or about 30 billion cells, at or about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject. In some embodiments, in the context of genetically engineered cells comprising the recombinant TCRs, a subject is administered at or about 10 million cells, at or about 100 million cells, at or about 1 billion cells, at or about 10 billion cells, at or about 100 billion cells, or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.

[0547] Dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments. In some embodiments, such values refer to numbers of recombinant receptor-expressing cells.

[0548] In some embodiments, the dose of genetically engineered T cells comprises between at or about 3 x 10 ⁷ recombinant TCR-expressing T cells and at or about 3 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive. In some embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR-expressing T cells and at or about 1 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive. In some embodiments, the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR- expressing T cells and at or about 1 x 10 ⁹ recombinant TCR-expressing T cells, inclusive.

[0549] In some embodiments, the dose of genetically engineered T cells comprises: at or about 1 x 10 ⁸ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR- expressing T cells; at or about 1 x 10 ⁹ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR-expressing T cells; or at or about 1 x IO ¹⁰ recombinant TCR-expressing T cells.

[0550] In some embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10 ⁸ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10 ⁸ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 1 x 10 ⁹ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises at or about 3 x 10 ⁹ recombinant TCR-expressing T cells. In some embodiments, dose of genetically engineered T cells comprises at or about 1 x 10 ¹⁰ recombinant TCR-expressing T cells. In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and/or CD8+ T cells.

[0551] In some embodiments, for example, where the subject is a human, the dose includes fewer than about 3 x 10 ¹¹ total recombinant TCR-expressing cells, e.g., in the range of from at or about 1 x 10 ⁶ to at or about 1.5 x 10 ¹¹ total of such cells, such as at or about 1 x 10 ⁷ , 3 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ , 1.25 x 10 ¹¹ , 2 x 10 ¹¹ total such cells, or the range between any two of the foregoing values. In some embodiments, for example, where the subject is a human, the dose includes more than at or about 1 x 10 ⁷ total recombinant TCR- expressing cells, and fewer than at or about 1 x 10 ¹¹ total recombinant TCR-expressing cells, e.g., in the range of at or about 1 x 10 ⁷ to at or about 1 x 10 ¹¹ such cells, such as at or about 5 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 1 x 10 ⁹ , 1 x 10 ¹⁰ , 5 x 10 ¹⁰ , 7.5 x 10 ¹⁰ , 1 x 10 ¹¹ total of such cells, or the range between any two of the foregoing values.

[0552] In some embodiments, the dose of genetically engineered cells comprises from at or about 1 x 10 ⁶ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1.5 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 5 x 10 ¹⁰ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1 x 10 ¹⁰ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1 x 10 ⁹ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 5 x 10 ⁸ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1 x 10 ⁸ total TCR- expressing cells, from at or about 1 x 10 ⁶ to at or about 5 x 10 ⁷ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 1 x 10 ⁷ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 5 x 10 ⁶ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 2.5 x 10 ⁶ total TCR-expressing cells, from at or about 1 x 10 ⁶ to at or about 2 x 10 ⁶ total TCR-expressing cells, from at or about 2 x 10 ⁶ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 2.5 x 10 ⁶ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 5 x 10 ⁶ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁷ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 3 x 10 ⁷ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁸ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 5 x 10 ⁸ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x 10 ⁹ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1 x IO ¹⁰ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 5 x IO ¹⁰ to at or about 2 x 10 ¹¹ total TCR- expressing cells, from at or about 1 x 10 ⁶ to at or about 2 x 10 ¹¹ total TCR-expressing cells, from at or about 1.5 x IO ¹⁰ to at or about 2 x 10 ¹¹ total TCR-expressing cells. In some embodiments, the dose of genetically engineered cells comprises from or from about 1 x 10 ⁷ to at or about 1 x 10 ¹¹ total TCR-expressing cells, such as from or from about 1 x 10 ⁹ to or to about 1 x IO ¹⁰ total TCR-expressing cells.

[0553] In some embodiments, the dose of genetically engineered cells comprises less than at or about 2 x 10 ¹¹ TCR-expressing cells, less than at or about 1.75 x 10 ¹¹ TCR-expressing cells, less than at or about 1.5 x 10 ¹¹ TCR-expressing cells, less than at or about 1.25 x 10 ¹¹ TCR- expressing cells, less than at or about 1 x 10 ¹¹ TCR-expressing cells, less than at or about 7.5 x 10 ¹⁰ TCR-expressing cells, less than at or about 5 x 10 ¹⁰ TCR-expressing cells, less than at or about 2.5 x 10 ¹⁰ TCR-expressing cells, less than at or about 1 x 10 ¹⁰ TCR-expressing cells, less than at or about 5 x 10 ⁹ TCR-expressing cells, less than at or about 1 x 10 ⁹ TCR-expressing cells, less than at or about 5 x 10 ⁸ TCR-expressing cells, less than at or about 6 x 10 ⁷ TCR- expressing cells, less than at or about 3 x 10 ⁷ TCR-expressing cells, less than at or about 1 x 10 ⁷ TCR-expressing cells, less than at or about 5 x 10 ⁶ TCR-expressing cells, less than at or about 1 x 10 ⁶ TCR-expressing cells.

[0554] In some embodiments, the dose of genetically engineered cells comprises at or about 3 x 10 ¹¹ TCR-expressing cells, at or about 2 x 10 ¹¹ TCR-expressing cells, at or about 1.75 x 10 ¹¹ TCR-expressing cells, at or about 1.5 x 10 ¹¹ TCR-expressing cells, at or about 1.25 x 10 ¹¹ TCR- expressing cells, at or about 1 x 10 ¹¹ TCR-expressing cells, at or about 7.5 x 10 ¹⁰ TCR- expressing cells, at or about 5 x 10 ¹⁰ TCR-expressing cells, at or about 2.5 x 10 ¹⁰ TCR- expressing cells, at or about 1 x 10 ¹⁰ TCR-expressing cells, at or about 5 x 10 ⁹ TCR-expressing cells, at or about 1 x 10 ⁹ TCR-expressing cells, at or about 5 x 10 ⁸ TCR-expressing cells, at or about 1 x 10 ⁸ TCR-expressing cells, at or about 5 x 10 ⁷ TCR-expressing cells, at or about 3 x 10 ⁷ TCR-expressing cells, at or about 1 x 10 ⁷ TCR-expressing cells, at or about 5 x 10 ⁶ TCR- expressing cells, at or about 1 x 10 ⁶ TCR-expressing cells. [0555] In some embodiments, the dose of cells comprises between at or about 2 x 10 ⁵ of the cells/kg and at or about 2 x 10 ⁶ of the cells/kg, such as between at or about 4 x 10 ⁵ of the cells/kg and at or about 1 x 10 ⁶ of the cells/kg or between at or about 6 x 10 ⁵ of the cells/kg and at or about 8 x 10 ⁵ of the cells/kg. In some embodiments, the dose of cells comprises no more than 2 x 10 ⁵ of the cells (e.g. recombinant TCR-expressing cells) per kilogram body weight of the subject (cells/kg), such as no more than at or about 3 x 10 ⁵ cells/kg, no more than at or about 4 x 10 ⁵ cells/kg, no more than at or about 5 x 10 ⁵ cells/kg, no more than at or about 6 x 10 ⁵ cells/kg, no more than at or about 7 x 10 ⁵ cells/kg, no more than at or about 8 x 10 ⁵ cells/kg, no more than at or about 9 x 10 ⁵ cells/kg, no more than at or about 1 x 10 ⁶ cells/kg, or no more than at or about 2 x 10 ⁶ cells/kg. In some embodiments, the dose of cells comprises at least at or about 2 x 10 ⁵ of the cells (e.g. recombinant TCR-expressing cells) per kilogram body weight of the subject (cells/kg), such as at least at or about 3 x 10 ⁵ cells/kg, at least at or about 4 x 10 ⁵ cells/kg, at least at or about 5 x 10 ⁵ cells/kg, at least at or about 6 x 10 ⁵ cells/kg, at least at or about 7 x 10 ⁵ cells/kg, at least at or about 8 x 10 ⁵ cells/kg, at least at or about 9 x 10 ⁵ cells/kg, at least at or about 1 x 10 ⁶ cells/kg, or at least at or about 2 x 10 ⁶ cells/kg.

[0556] In some embodiments, the populations or sub-types of cells, such as CD8 ⁺ and CD4 ⁺ T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4 ⁺ to CD8 ⁺ ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0557] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight.

[0558] Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 ⁺ to CD8 ⁺ cells, and/or is based on a desired fixed or minimum dose of CD4 ⁺ and/or CD8 ⁺ cells.

[0559] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4 ⁺ and CD8 ⁺ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4 ⁺ to CD8 ⁺ cells) is between at or about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or about 5:l, 4.5:1, 4:1, 3.5:1, 3: 1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0560] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and/or the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose.

[0561] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose.

[0562] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is at or about 50% of the total cells in the dose; and the percentage of CD8+ T cells in the dose is at or about 50% of the total cells in the dose.

[0563] In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1. In some embodiments, the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is at or about 1:1.

[0564] In particular embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0565] In some embodiments, the dose of cells, e.g., recombinant TCR-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more. In some embodiments, the subject is administered one or more doses.

[0566] In some embodiments, the recombinant TCRs or cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another TCR, antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.

[0567] The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.

[0568] Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or recombinant TCRs in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. [0569] In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered TCRs or antibody-expressing CARs expressed by the engineered cells in some embodiments are conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the TCR or CAR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Patent 5,087,616.

V. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0570] Also provided are compositions, such as pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. In some aspects, the pharmaceutical compositions contain any of the engineered cells or compositions containing the engineered cells described herein, e.g., engineered to express a recombinant TCR. In some embodiments, the dose of cells comprising the provided engineered cells, e.g., engineered to express a recombinant TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accord with the provided methods, and/or with the provided articles of manufacture or compositions, such as in the prevention or treatment of diseases, conditions, and disorders, or in detection, diagnostic, and prognostic methods.

[0571] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0572] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0573] In some aspects, the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0574] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0575] The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being prevented or treated with the cells or agents, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agents or cells are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.

[0576] The pharmaceutical composition in some embodiments contains agents or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactic ally effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0577] The agents or cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells or agent. In some embodiments, it is administered by multiple bolus administrations of the cells or agent, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells or agent.

[0578] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or therapeutic purposes, previous therapy, the subject’s clinical history and response to the agent or the cells, and the discretion of the attending physician. The compositions are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0579] The cells or agents may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. With respect to cells, administration can be autologous or heterologous. In some aspects, the cells are isolated from a subject, engineered, and administered to the same subject. In other aspects, they are isolated from one subject, engineered, and administered to another subject. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell or an agent that treats or ameliorates symptoms of neurotoxicity), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0580] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0581] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0582] Sterile injectable solutions can be prepared by incorporating the agent or cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0583] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

VI. KITS AND ARTICLES OF MANUFACTURE

[0584] Also provided are articles of manufacture, systems, apparatuses, and kits useful in performing the provided embodiments. In some embodiments, the provided articles of manufacture or kits contain one or more components of the one or more agent(s) capable of inducing genetic disruption and/or polynucleotide(s), e.g., template polynucleotides containing transgene sequences encoding a recombinant receptor or a portion thereof. In some embodiments, the articles of manufacture or kits can be used in methods for engineering T cells to express a recombinant TCR and/or other molecules as described herein, for example, to generate the engineered cells comprising a genetic disruption at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus and a modified TRAC locus comprising a transgene encoding a recombinant TCR or a portion thereof.

[0585] In some embodiments, the articles of manufacture or kits include polypeptides, nucleic acids, vectors and/or polynucleotides useful in performing the provided methods. In some embodiments, the articles of manufacture or kits include one or more agent(s) capable of inducing a genetic disruption, for example, at a TGFBR2 locus and/or a TRAC locus (such as those described in Section LA herein). In some embodiments, the articles of manufacture or kits include one or more nucleic acid molecules, e.g., a plasmid or a DNA fragment, that encodes one or more components of the one or more agent(s) capable of inducing genetic disruption and/or comprises polynucleotide(s), e.g., template polynucleotides for use in targeting transgene sequences into the cell via HDR, such as those described in Section I.B.2 herein. In some embodiments, the articles of manufacture or kits provided herein contain control vectors.

[0586] In some embodiments, the articles of manufacture or kits provided herein contain one or more agent(s), wherein each of the one or more agent is independently capable of inducing a genetic disruption of a target site within a TGFBR2 locus and a TRAC locus; and a polynucleotide comprising a transgene encoding a recombinant receptor or a portion thereof, wherein the transgene is targeted for integration at or near the target site via homology directed repair (HDR). In some aspects, the one or more agent(s) capable of inducing a genetic disruption is any described herein. In some aspects, the one or more agent(s) is a ribonucleoprotein (RNP) complex comprising a Cas9/gRNA complex. In some aspects, the gRNA included in the RNP targets a target site in the TGFBR2 locus or a TRAC locus or both, such as any first target site and/or a second target site described herein. In some aspects, the template polynucleotide is any of the template polynucleotide described herein.

[0587] In some embodiments, the articles of manufacture or kits include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and/or packaging, generally including instructions for use, e.g., instructions for introducing the components into the cells for engineering.

[0588] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging the provided materials are well known. See, for example, U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies, e.g., pipette tips and/or plastic plates, or bottles. The articles of manufacture or kits can include a device so as to facilitate dispensing of the materials or to facilitate use in a high-throughput or large-scale manner, e.g., to facilitate use in robotic equipment. Typically, the packaging is non- reactive with the compositions contained therein.

[0589] In some embodiments, the one or more agent(s) capable of inducing genetic disruption and/or template polynucleotide(s) are packaged separately. In some embodiments, each container can have a single compartment. In some embodiments, other components of the articles of manufacture or kits are packaged separately, or together in a single compartment.

[0590] Also provided are articles of manufacture, systems, apparatuses, and kits useful in administering the provided cells and/or cell compositions, e.g., for use in therapy or treatment. In some embodiments, the articles of manufacture or kits provided herein contain T cells and/or T cell compositions, such as any T cells and/or T cell compositions described herein. In some aspects, the articles of manufacture or kits provided herein can be used for administration of the T cells or T cell compositions, and can include instructions for use.

[0591] In some embodiments, the articles of manufacture or kits provided herein contain T cells, and/or T cell compositions, such as any T cells, and/or pharmaceutical compositions comprising T cells described herein. In some embodiments, the T cells, and/or Pharmaceutical compositions any of the modified T cells used the screening methods described herein. In some embodiments, the articles of manufacture or kits provided herein contain control or unmodified T cells and/or Pharmaceutical compositions. In some embodiments, the article of manufacture or kits include one or more instructions for administration of the engineered cells and/or cell compositions for therapy.

[0592] The articles of manufacture and/or kits containing cells or cell compositions for therapy, may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection, or bottles or vials for orally administered agents. The label or package insert may indicate that the composition is used for treating a disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes engineered cells expressing a recombinant receptor; and (b) a second container with a composition contained therein, wherein the composition includes the second agent. In some embodiments, the article of manufacture may include (a) a first container with a first composition contained therein, wherein the composition includes a subtype of engineered cells expressing a recombinant receptor; and (b) a second container with a composition contained therein, wherein the composition includes a different subtype of engineered cells expressing a recombinant receptor. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.

VII. DEFINITIONS

[0593] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0594] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of’ aspects and variations.

[0595] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0596] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In some embodiments, “about” may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%.

[0597] As used herein, recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[0598] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as retroviral, e.g., gammaretroviral and lentiviral vectors.

[0599] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0600] “Isolated nucleic acid encoding a TCR or an antibody” refers to one or more nucleic acid molecules encoding TCR alpha or P chains (or fragments thereof) or antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0601] " Domain" is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

[0602] The term "variant" refers to an entity such as a polypeptide, polynucleotide or small molecule that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity.

[0603] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0604] As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

[0605] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0606] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, in some embodiments, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0607] In some embodiments, “operably linked” may include the association of components, such as a DNA sequence, e.g. a heterologous nucleic acid) and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence. Hence, it means that the components described are in a relationship permitting them to function in their intended manner.

[0608] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a recombinant TCR of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity.

[0609] Amino acids generally can be grouped according to the following common sidechain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0610] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0611] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0612] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

[0613] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

VIII. EXEMPLARY EMBODIMENTS

[0614] Among the provided embodiments are:

1. A genetically engineered T cell, comprising: a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, and a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; wherein: the genetically engineered T cells comprises a modified T cell receptor alpha constant (TRAC) locus comprising a transgene encoding the recombinant TCR or portion thereof; and reduced expression of the endogenous TGFBR2 locus.

2. A genetically engineered T cell, comprising: a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, and a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; wherein: the genetically engineered T cells comprises a modified T cell receptor alpha constant (TRAC) locus comprising a transgene encoding the TCRa chain and the TCRP chain of the recombinant TCR; and reduced expression of the endogenous TGFBR2 locus.

3. The genetically engineered T cell of embodiment 1 or 2, wherein the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus.

4. The genetically engineered T cell of any of embodiments 1-3, wherein the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus.

5. The genetically engineered T cell of any of embodiments 1-4, wherein the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

6. The genetically engineered T cell of any of embodiments 1-5, wherein: the genetically engineered T cell does not encode a functional TGFBR2 polypeptide; the genetically engineered T cell does not encode a TGFBR2 polypeptide; the genetically engineered T cell does not encode a full length TGFBR2 polypeptide; the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell; and/or

TGFP signal transmission is reduced or eliminated in the genetically engineered T cell.

7. The genetically engineered T cell of any of embodiments 1-6, wherein the transgene has been integrated via homology directed repair (HDR) at the TRAC locus in a cell comprising a second genetic disruption at a second target site at an endogenous TRAC locus.

8. The genetically engineered T cell of embodiment 7, wherein the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

9. The genetically engineered T cell of embodiment 7 or 8 wherein: the first genetic disruption has been introduced using a first agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination; and/or the second genetic disruption has been introduced using a second agent comprising a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination.

10. The genetically engineered T cell of embodiment 9, wherein: the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA

(gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein; and/or the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein.

11. The genetically engineered T cell of embodiment 10, wherein the Cas9 protein is a S. pyogenes Cas9 protein.

12. The genetically engineered T cell of any of embodiments 1-4 and 6-9, wherein the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129.

13. The genetically engineered T cell of any of embodiments 1-12, wherein the first target site comprises the sequence of SEQ ID NO:83.

14. The genetically engineered T cell of embodiment 13, wherein the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG).

15. The genetically engineered T cell of any of embodiments 7-14, wherein the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265.

16. The genetically engineered T cell of embodiment 15, wherein the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55.

17. The genetically engineered T cell of any of embodiments 7-15, wherein the second target site comprises the sequence of SEQ ID NO:238.

18. The genetically engineered T cell of embodiment 17, wherein the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

19. The genetically engineered T cell of any of embodiments 7-18, wherein the integration of the transgene via HDR is carried out with a polynucleotide comprising the structure [5’ homology arm] -[transgene] -[3’ homology arm].

20. The genetically engineered T cell of embodiment 19, wherein the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site.

21. The genetically engineered T cell of embodiment 19 or 20, wherein the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof.

22. The genetically engineered T cell of any of embodiments 19-21, wherein the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

23. The genetically engineered T cell of any of embodiments 1-22, wherein the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV).

24. The genetically engineered T cell of any of embodiments 1-23, wherein the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l l-19) YMLDLQPET (SEQ ID NO: 267), optionally wherein the MHC molecule is an HLA-A2 molecule.

25. The genetically engineered T cell of any of embodiments 1-24, wherein: the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR- 3 comprising the sequence of SEQ ID NO:5.

26. The genetically engineered T cell of any of embodiments 1-25, wherein: the Va region comprise the sequence of SEQ ID NO:8, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

27. The genetically engineered T cell of any of embodiments 1-26, wherein: the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

28. The genetically engineered T cell of any of embodiments 1-27, wherein the TCRa chain comprises a constant alpha (Ca) region and the TCRP chain comprises a constant beta (CP) region.

29. The genetically engineered T cell of embodiment 28, wherein the Ca region and the CP region are human constant regions.

30. The genetically engineered T cell of embodiment 28 or 29, wherein: the Ca region comprises a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region comprises a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183. 31. The genetically engineered T cell of any of embodiments 28-30, wherein the Ca region and the CP region comprise one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

32. The genetically engineered T cell of any of embodiments 28-31, wherein: the Ca region comprises the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2.

33. The genetically engineered T cell of any of embodiments 28-32, wherein: the Ca region comprises the sequence of SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2.

34. The genetically engineered T cell of any of embodiments 1-33, wherein: the TCRa chain comprises the sequence of SEQ ID NO: 14; and the TCRP chain comprises the sequence of SEQ ID NO:7.

35. The genetically engineered T cell of any of embodiments 1-34, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

36. The genetically engineered T cell of any of embodiments 1-35, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO:18.

37. The genetically engineered T cell of any of embodiments 1-36, wherein the transgene comprises a nucleotide sequence encoding at least one further protein, optionally wherein the at least one further protein comprises a surrogate marker, optionally wherein the surrogate marker is a truncated receptor, optionally wherein the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand. 38. The genetically engineered T cell of any of embodiments 1-37, wherein the transgene comprises one or more multicistronic element(s).

39. The genetically engineered T cell of embodiment 38, wherein: the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain; and/or the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

40. The genetically engineered T cell of embodiment 38 or 39, wherein the one or more multicistronic element is or comprises a ribosome skip sequence, optionally wherein the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element.

41. The genetically engineered T cell of any of embodiments 38-40, wherein the one or more multicistronic element comprises a P2A element.

42. The genetically engineered T cell of embodiment 41, wherein the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234, optionally SEQ ID NO:233.

43. The genetically engineered T cell of any of embodiments 1-42, wherein the transgene comprises the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24.

44. The genetically engineered T cell of any of embodiments 1-43, wherein the transgene comprises the sequence of SEQ ID NO:24.

45. The genetically engineered T cell of any of embodiments 1-44, wherein the transgene comprises one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell.

46. The genetically engineered T cell of embodiment 45, wherein the heterologous regulatory or control element comprises a heterologous promoter.

47. The genetically engineered T cell of embodiment 46, wherein the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

48. The genetically engineered T cell of any of embodiments 1-47, wherein the genetically engineered T cell: is less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), optionally immune suppression mediated by TGFp, when administered to a subject having a disease or disorder; results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder; results in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder; exhibits a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR; results in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder; and/or results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

49. The genetically engineered T cell of embodiment 48, wherein the disease or disorder is associated with HPV, optionally HPV 16.

50. The genetically engineered T cell of embodiment 48 or 49, wherein the disease or disorder is a cancer or a tumor, optionally a solid tumor.

51. The genetically engineered T cell of any of embodiments 1-50, wherein the T cell is a primary T cell derived from a subject, optionally wherein the subject is a human.

52. The genetically engineered T cell of any of embodiments 1-51, wherein the T cell is a CD8+ T cell or subtypes thereof.

53. The genetically engineered T cell of any of embodiments 1-51, wherein the T cell is a CD4+ T cell or subtypes thereof.

54. A method of producing a genetically engineered T cell, the method comprising:

(a) introducing, into a T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; and

(b) introducing a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus; and

(c) introducing a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof.

55. A method of producing a genetically engineered T cell, the method comprising: (a) introducing, into a T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus; and

(b) introducing a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus; and

(c) introducing a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region.

56. A method of producing a genetically engineered T cell, the method comprising introducing, into a T cell, a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof, said T cell comprising a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

57. A method of producing a genetically engineered T cell, the method comprising introducing, into a T cell, a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, said T cell comprising a first genetic disruption at a first target site within an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

58. The method of embodiment 56 or 57, wherein the first genetic disruption and the second genetic disruption is carried out by introducing, into the T cell, a first agent for inducing a first genetic disruption at a first target site within an endogenous transforming growth factorbeta receptor type-2 (TGFBR2) locus and a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus.

59. The method of any of embodiments 54-58, wherein the polynucleotide further comprises one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of a TRAC locus.

60. The method of any of embodiments 54-59, wherein the transgene is integrated via homology directed repair (HDR) at the TRAC locus.

61. The method of any of embodiments 54-60, wherein the method thereby produces a genetically engineered T cell comprising: a modified T cell receptor alpha constant (TRAC) locus comprising the transgene encoding the recombinant T cell receptor (TCR) or portion thereof; and the first genetic disruption at the first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and reduced expression of the endogenous TGFBR2 locus.

62. The method of any of embodiments 54-61, wherein the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus.

63. The method of any of embodiments 54-62, wherein the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus.

64. The method of any of embodiments 54-63, wherein the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

65. The method of any of embodiments 54-64, wherein: the genetically engineered T cell produced by the method does not encode a functional TGFBR2 polypeptide; the genetically engineered T cell produced by the method does not encode a TGFBR2 polypeptide; the genetically engineered T cell produced by the method does not encode a full length TGFBR2 polypeptide; the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell produced by the method; and/or

TGFP signal transmission is reduced or eliminated in the genetically engineered T cell produced by the method.

66. The method of any of embodiments 54-65, wherein the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

67. The method of any of embodiments 54, 55, and 58-66, wherein: the first agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination; and/or the second agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease

(TALEN), or a CRISPR-Cas combination. 68. The method of embodiment 67, wherein: the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein; and/or the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein.

69. The method of embodiment 68, wherein the Cas9 protein is a S. pyogenes Cas9 protein.

70. The method of any of embodiments 54-69, wherein the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129.

71. The method of any of embodiments 54-70, wherein the first target site comprises the sequence of SEQ ID NO:83.

72. The method of embodiment 71, wherein the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG).

73. The method of any of embodiments 54-72, wherein the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265.

74. The method of embodiment 73, wherein the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55.

75. The method of any of embodiments 54-73, wherein the second target site comprises the sequence of SEQ ID NO:238.

76. The method of embodiment 75, wherein the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

77. The method of any of embodiments 54-76, wherein the polynucleotide comprises the structure [5’ homology arm] -[transgene] -[3’ homology arm].

78. The method of embodiment 77, wherein the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site.

79. The method of embodiment 77 or 78, wherein the 5’ homology arm and 3’ homology arm independently are at or about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the foregoing, or are greater than at or about 300 nucleotides in length, optionally at or about 400, 500 or 600 nucleotides in length.

80. The method of any of embodiments 77-79, wherein the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof.

81. The method of any of embodiments 77-80, wherein the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

82. The method of any of embodiments 54-81, wherein the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV).

83. The method of any of embodiments 54-82, wherein the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l l-19) YMLDLQPET (SEQ ID NO: 267), optionally wherein the MHC molecule is an HLA-A2 molecule.

84. The method of any of embodiments 54-83, wherein: the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR- 3 comprising the sequence of SEQ ID NO:5.

85. The method of any of embodiments 54-84, wherein: the Va region comprise the sequence of SEQ ID NO:8, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

86. The method of any of embodiments 54-85, wherein: the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

87. The method of any of embodiments 54-86, wherein the TCRa chain comprises a constant alpha (Ca) region and the TCRP chain comprises a constant beta (CP) region.

88. The method of embodiment 87, wherein the Ca region and the CP region are human constant regions.

89. The method of embodiment 87 or 88, wherein: the Ca region comprises a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region comprises a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183.

90. The method of any of embodiments 87-89, wherein the Ca region and the CP region comprise one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

91. The method of any of embodiments 87-90, wherein: the Ca region comprises the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2.

92. The method of any of embodiments 87-91, wherein: the Ca region comprises the sequence of SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2.

93. The method of any of embodiments 54-92, wherein: the TCRa chain comprises the sequence of SEQ ID NO: 14; and the TCRP chain comprises the sequence of SEQ ID NO:7.

94. The method of any of embodiments 54-93, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

95. The method of any of embodiments 54-94, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO:18.

96. The method of any of embodiments 54-95, wherein the transgene comprises a nucleotide sequence encoding at least one further protein, optionally wherein the at least one further protein comprises a surrogate marker, optionally wherein the surrogate marker is a truncated receptor, optionally wherein the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand.

97. The method of any of embodiments 54-96, wherein the transgene comprises one or more multicistronic element(s).

98. The method of embodiment 97, wherein: the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain; and/or the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

99. The method of embodiment 97 or 98, wherein the one or more multicistronic element is or comprises a ribosome skip sequence, optionally wherein the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element.

100. The method of any of embodiments 97-99, wherein the one or more multicistronic element comprises a P2A element.

101. The method of embodiment 100, wherein the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234, optionally SEQ ID NO:233.

102. The method of any of embodiments 54-101, wherein the transgene comprises the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24.

103. The method of any of embodiments 54-102, wherein the transgene comprises the sequence of SEQ ID NO:24.

104. The method of any of embodiments 54-103, wherein the transgene comprises one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell.

105. The method of embodiment 104, wherein the heterologous regulatory or control element comprises a heterologous promoter.

106. The method of embodiment 105, wherein the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

107. The method of any of embodiments 54-106, wherein the polynucleotide is comprised in a viral vector. 108. The method of embodiment 107, wherein the viral vector is an AAV vector, optionally wherein the AAV vector is an AAV6 vector.

109. The method of embodiment 107, wherein the viral vector is a retroviral vector, optionally a lentiviral vector.

110. The method of any of embodiments 54-106, wherein the polynucleotide is a linear polynucleotide, optionally a double-stranded polynucleotide or a single-stranded polynucleotide.

111. The method of any of embodiments 54-110, wherein the polynucleotide is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length.

112. A system for engineering a T cell, comprising:

(a) a first agent for inducing a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus of a T cell;

(b) a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus of the T cell; and

(c) a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region, or a portion thereof; and one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of the TRAC locus.

113. A system for engineering a T cell, comprising:

(a) a first agent for inducing a first genetic disruption at a first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus of a T cell;

(b) a second agent for inducing a second genetic disruption at a second target site within a T cell receptor alpha constant (TRAC) locus of the T cell; and

(c) a polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) comprising a TCR alpha (TCRa) chain comprising a variable alpha (Va) region, and a TCR beta (TCRP) chain comprising a variable beta (VP) region; and one or more homology arm(s) linked to the transgene, wherein the one or more homology arm(s) comprise a sequence homologous to one or more region(s) of an open reading frame of the TRAC locus.

114. The system of embodiment 112 or 113, wherein the transgene is a sequence that is exogenous or heterologous to the T cell.

115. The system of any of embodiments 112-114, wherein the first agent, the second agent and the polynucleotide are for introduction into the T cell, and the transgene is integrated via homology directed repair (HDR) at the TRAC locus.

116. The system of embodiment 115, wherein the introduction of the first agent, the second agent and the polynucleotide into the T cell produces a genetically engineered T cell comprising: a modified T cell receptor alpha constant (TRAC) locus comprising the transgene encoding the recombinant T cell receptor (TCR) or portion thereof; and the first genetic disruption at the first target site at an endogenous transforming growth factor-beta receptor type-2 (TGFBR2) locus, and reduced expression of the endogenous TGFBR2 locus.

117. The system of any of embodiments 112-116, wherein the first target site is present at a promoter, a regulatory element or a control element of the endogenous TGFBR2 locus or an open reading frame of the endogenous TGFBR2 locus.

118. The system of any of embodiments 112-117, wherein the first target site is present downstream of exon 1 and upstream of exon 8 of an open reading frame of the endogenous TGFBR2 locus.

119. The system of any of embodiments 112-118, wherein the first target site is present downstream of exon 4 and upstream of exon 6, of the open reading frame of the endogenous TGFBR2 locus.

120. The system of any of embodiments 112-119, wherein: the genetically engineered T cell does not encode a functional TGFBR2 polypeptide; the genetically engineered T cell does not encode a TGFBR2 polypeptide; the genetically engineered T cell does not encode a full length TGFBR2 polypeptide; the expression of TGFBR2 polypeptide is reduced or eliminated in the genetically engineered T cell; and/or

TGFP signal transmission is reduced or eliminated in the genetically engineered T cell.

121. The system of any of embodiments 112-120, wherein the second target site is present at a promoter, a regulatory element or a control element of the endogenous TRAC locus or an open reading frame of the endogenous TRAC locus.

122. The system of any of embodiments 112-121, wherein: the first agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination; and/or the second agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination.

123. The system of embodiment 122, wherein: the first agent comprises a first CRISPR-Cas combination comprising a first guide RNA (gRNA) comprising a first targeting domain that binds to the first target site, and a Cas9 protein; and/or the second agent comprises a second CRISPR-Cas combination comprising a second gRNA comprising a second targeting domain that binds to the second target site, and a Cas9 protein.

124. The system of embodiment 123, wherein: the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein; and the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

125. The system of embodiment 123 or 124, wherein the Cas9 protein is a S. pyogenes Cas9 protein.

126. The system of any of embodiments 112-118 and 120-125, wherein the first target site comprises a sequence selected from among any one of SEQ ID NOS:59-129.

127. The system of any of embodiments 112-126, wherein the first target site comprises the sequence of SEQ ID NO: 83.

128. The system of embodiment 127, wherein the first targeting domain comprises the sequence of SEQ ID NO:58 (GUGGAUGACCUGGCUAACAG).

129. The system of any of embodiments 112-128, wherein the second target site comprises a sequence selected from among any one of SEQ ID NOS:235-265.

130. The system of any of embodiments 112-129, wherein the second targeting domain comprises a sequence selected from among any one of SEQ ID NOS:25-55.

131. The system of any of embodiments 112-130, wherein the second target site comprises the sequence of SEQ ID NO:238.

132. The system of embodiment 131, wherein the second targeting domain comprises the sequence of SEQ ID NO:28 (GAGAAUCAAAAUCGGUGAAU).

133. The system of any of embodiments 112-132, wherein the polynucleotide comprises the structure [5’ homology arm] -[transgene] -[3’ homology arm].

134. The system of embodiment 133, wherein the 5’ homology arm and 3’ homology arm comprises nucleic acid sequences homologous to nucleic acid sequences surrounding the second target site.

135. The system of embodiment 133 or 134, wherein the 5’ homology arm and 3’ homology arm independently are at or about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the foregoing, or are greater than at or about 300 nucleotides in length, optionally at or about 400, 500 or 600 nucleotides in length.

136. The system of any of embodiments 133-135, wherein the 5’ homology arm comprises SEQ ID NO: 56 or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56 or a partial sequence thereof, and/or the 3’ homology arm comprises SEQ ID NO:57, a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57 or a partial sequence thereof.

137. The system of any of embodiments 133-136, wherein the 5’ homology arm comprises SEQ ID NO: 56 and the 3’ homology arm comprises SEQ ID NO:57.

138. The system of any of embodiments 112-137, wherein the recombinant TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV).

139. The system of any of embodiments 112-138, wherein the recombinant TCR binds to or recognizes HPV 16 E7 in the context of an MHC molecule, wherein the peptide epitope is or comprises E7(l 112-19) YMLDLQPET (SEQ ID NO: 267), optionally wherein the MHC molecule is an HLA-A2 molecule.

140. The system of any of embodiments 112-139, wherein: the Va region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO: 10, a CDR-2 comprising the sequence of SEQ ID NO: 11, and a CDR-3 comprising the sequence of SEQ ID NO: 12; and the VP region comprises a complementarity determining region 1 (CDR-1) comprising the sequence of SEQ ID NO:3, a CDR-2 comprising the sequence of SEQ ID NO:4, and a CDR- 3 comprising the sequence of SEQ ID NO:5.

141. The system of any of embodiments 112-140, wherein: the Va region comprise the sequence of SEQ ID NO:8, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

142. The system of any of embodiments 112-141, wherein: the Va region comprise the sequence of SEQ ID NO:8; and the VP region comprise the sequence of SEQ ID NO:1.

143. The system of any of embodiments 112-142, wherein the TCRa chain comprises a constant alpha (Ca) region and the TCRP chain comprises a constant beta (CP) region.

144. The system of embodiment 143, wherein the Ca region and the CP region are human constant regions.

145. The system of embodiment 143 or 144, wherein: the Ca region comprises a sequence selected from any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 9, 167-172, 175, 176, and 178-181; and the CP region comprises a sequence selected from any one of SEQ ID NOS:2, 156, 173, 174, 177, 182, and 183, or a sequence that has at least at or about 90% sequence identity to any one of SEQ ID NOS: 2, 156, 173, 174, 177, 182, and 183.

146. The system of any of embodiments 143-145, wherein the Ca region and the CP region comprise one or more modifications comprising cysteine residues that are capable of forming one or more non-native disulfide bridges between the TCRa chain and TCRP chain.

147. The system of any of embodiments 143-146, wherein: the Ca region comprises the sequence of SEQ ID NO:9, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2, or a sequence that has at least at or about 90% sequence identity to SEQ ID NO:2.

148. The system of any of embodiments 143-147, wherein: the Ca region comprises the sequence of SEQ ID NO:9; and the CP region comprises the sequence of SEQ ID NO:2.

149. The system of any of embodiments 112-148, wherein: the TCRa chain comprises the sequence of SEQ ID NO: 14; and the TCRP chain comprises the sequence of SEQ ID NO:7.

150. The system of any of embodiments 112-149, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID NO:22, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID NO: 18, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

151. The system of any of embodiments 112-150, wherein the transgene comprises: a nucleotide sequence encoding the TCRa chain comprising the sequence of SEQ ID

NO:22; and a nucleotide sequence encoding the TCRP chain comprising the sequence of SEQ ID

NO:18.

152. The system of any of embodiments 112-151, wherein the transgene comprises a nucleotide sequence encoding at least one further protein, optionally wherein the at least one further protein comprises a surrogate marker, optionally wherein the surrogate marker is a truncated receptor, optionally wherein the truncated receptor lacks an intracellular signaling domain and/or is not capable of mediating intracellular signaling when bound by its ligand.

153. The system of any of embodiments 112-152, wherein the transgene comprises one or more multicistronic element(s).

154. The system of embodiment 153, wherein: the one or more multicistronic element(s) are positioned between the nucleotide sequence encoding the TCRa chain and the nucleotide sequence encoding the TCRP chain; and/or the one or more multicistronic element(s) are positioned upstream of the nucleotide sequence encoding the recombinant TCR or a portion thereof; and/or are positioned between the nucleotide sequence encoding the recombinant TCR or a portion thereof and the nucleotide sequence encoding the at least one further protein.

155. The system of embodiment 153 or 154, wherein the one or more multicistronic element is or comprises a ribosome skip sequence, optionally wherein the ribosome skip sequence is a T2A, a P2A, an E2A, or an F2A element.

156. The system of any of embodiments 153-155, wherein the one or more multicistronic element comprises a P2A element.

157. The system of embodiment 156, wherein the P2A element comprises a sequence of any one of SEQ ID NOS: 221-234, optionally SEQ ID NO:233.

158. The system of any of embodiments 112-157, wherein the transgene comprises the sequence of SEQ ID NO:24, or a sequence that has at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24.

159. The system of any of embodiments 112-158, wherein the transgene comprises the sequence of SEQ ID NO:24.

160. The system of any of embodiments 112-159, wherein the transgene comprises one or more heterologous or regulatory control element(s) operably linked to control expression of the TCR when expressed from a cell introduced with the genetically engineered T cell.

161. The system of embodiment 160, wherein the heterologous regulatory or control element comprises a heterologous promoter. 162. The system of embodiment 161, wherein the heterologous promoter is or comprises a human elongation factor 1 alpha (EFla) promoter or a variant thereof.

163. The system of any of embodiments 112-162, wherein the polynucleotide is comprised in a viral vector.

164. The system of embodiment 163, wherein the viral vector is an AAV vector, optionally wherein the AAV vector is an AAV6 vector.

165. The system of embodiment 163, wherein the viral vector is a retroviral vector, optionally a lentiviral vector.

166. The system of any of embodiments 112-162, wherein the polynucleotide is a linear polynucleotide, optionally a double-stranded polynucleotide or a single-stranded polynucleotide.

167. The system of any of embodiments 112-166, wherein the polynucleotide is between at or about 2500 and at or about 5000 nucleotides, at or about 3500 and at or about 4500 nucleotides, or at or about 3750 nucleotides and at or about 4250 nucleotides in length.

168. The system of any of embodiments 112-167, wherein: the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein; and the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

169. The system of embodiment 168, wherein the concentration of the first RNP and/or the second RNP are between at or about 1 pM and at or about 5 pM, between at or about 1.5 pM and at or about 2.5 pM, between at or about 1.7 pM and at or about 2.5 pM, or between at or about 2 pM and at or about 2.5 pM, optionally at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM.

170. A method of producing a genetically engineered T cell, the method comprising introducing the first agent, the second agent and the polynucleotide of the system of any of embodiments 112-169, into a T cell.

171. The method of any of embodiments 54-111 and 170, wherein: the first agent comprises a first ribonucleoprotein (RNP) complex comprising the first gRNA and the Cas9 protein; and the second agent comprises a second RNP complex comprising the second gRNA and the Cas9 protein.

172. The method of embodiment 171, wherein the first RNP and/or the second RNP are introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing, optionally via electroporation. 173. The method of embodiment 171 or 172, wherein the concentration of the first RNP and/or the second RNP are between at or about 1 |aM and at or about 5 |aM, between at or about

I.5 |JM and at or about 2.5 |aM, between at or about 1.7 |aM and at or about 2.5 |aM, or between at or about 2 |aM and at or about 2.5 |aM, optionally at or about 1.0 pM, at or about 1.5 pM, at or about 1.7 pM, at or about 2 pM, at or about 2.2 pM, or at or about 2.5 pM.

174. The method of any of embodiments 54-111 and 170-173, wherein the first agent and the second agent are introduced simultaneously.

175. The method of any of embodiments 54-111 and 170-173, wherein the first agent and the second agent are introduced sequentially, in any order.

176. The method of any of embodiments 54-111 and 170-175, wherein the polynucleotide is introduced after the introduction of the first agent and/or the second agent.

177. The method of embodiment 176, wherein the polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of the agent.

178. The method of any of embodiments 54-111 and 170-177, wherein prior to the introducing of the first agent and/or the second agent, the method comprises incubating the T cells, in vitro with one or more stimulatory agent(s) under conditions to stimulate or activate the T cells, optionally wherein the one or more stimulatory agent(s) comprises anti-CD3 and/or anti- CD28 antibodies, optionally anti-CD3/anti-CD28 Fab conjugated oligomeric reagent.

179. The method of any of embodiments 54-111 and 170-178, wherein the method further comprises incubating the cells prior to, during or subsequent to the introducing of the first agent and/or the second agent and/or the introducing of the polynucleotide with one or more recombinant cytokines.

180. The method of embodiment 178 or 179, wherein the incubation is carried out subsequent to the introducing of the first agent and/or the second agent and the introducing of the polynucleotide for up to or approximately 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10,

I I, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to or about 7 days.

181. The method of any of embodiments 54-111 and 170-180, wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus.

182. The method of any of embodiments 54-111 and 170-181, wherein at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR.

183. The method of any of embodiments 54-111 and 170-182, wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method do not express a gene product of an endogenous TRAC locus.

184. The method of any of embodiments 54-111 and 170-183, wherein: at least at or about 80% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells, or of the total cells or total T cells, in a population containing a plurality of engineered T cells generated by the method do not express a gene product of an endogenous TRAC locus.

185. The method of any of embodiments 54-111 and 170-184, wherein the T cell is a primary T cell derived from a subject, optionally wherein the subject is a human.

186. The method of any of embodiments 54-111 and 170-185, wherein the T cell is a CD8+ T cell or subtypes thereof.

187. The method of any of embodiments 54-111 and 170-185, wherein the T cell is a CD4+ T cell or subtypes thereof.

188. The method of any of embodiments 54-111 and 170-187, wherein the genetically engineered T cell produced by the method: is less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), optionally immune suppression mediated by TGFp, when administered to a subject having a disease or disorder; results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder; results in increased modified tumor control index (mTCI) when administered to a subject having a disease or disorder; exhibits a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR; results in greater systemic expansion and/or longer persistence when administered to a subject having a disease or disorder; and/or results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to a subject having a disease or disorder at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

189. The method of embodiment 188, wherein the disease or disorder is associated with HPV, optionally HPV 16.

190. The method of embodiment 188 or 189, wherein the disease or disorder is a cancer or a tumor, optionally a solid tumor.

191. A genetically engineered T cell generated using the method of any of embodiments 54-111 and 170-190.

192. A composition, comprising the genetically engineered T cell any of embodiments 1-53 and 191.

193. A composition, comprising a plurality of the genetically engineered T cell of any of embodiments 1-53 and 191.

194. The composition of embodiment 192 or 193, further comprising a pharmaceutically acceptable excipient.

195. The composition of any of embodiments 192-194, wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in the composition comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus.

196. The composition of any of embodiments 192-195, wherein at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in the composition express the recombinant TCR and/or exhibits binding to the antigen recognized by the recombinant TCR.

197. The composition of any of embodiments 192-196, wherein at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered T cells, or of the total cells or total T cells, in the composition do not express a gene product of an endogenous TRAC locus.

198. The composition of any of embodiments 192-197, wherein: at least at or about 80% of the engineered T cells, or of the total cells or total T cells, in the composition comprise a genetic disruption at a first target site within an endogenous TGFBR2 locus; at least at or about 75% of the engineered T cells, or of the total cells or total T cells, in the composition express the recombinant TCR or exhibits binding to the antigen recognized by the recombinant TCR; and at least at or about 95% of the engineered T cells, or of the total cells or total T cells, in the composition do not express a gene product of an endogenous TRAC locus.

199. The composition of any of embodiments 192-198, wherein the composition comprises CD4+ T cells and/or CD8+ T cells.

200. The composition of any of embodiments 192-199, wherein the composition comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition; and/or the percentage of CD8+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition.

201. The composition of any of embodiments 192-200, wherein the composition comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1, optionally at or about 1:1.

202. A method of treating a disease or disorder, the method comprising administering the genetically engineered T cell any of embodiments 1-53 and 191, a plurality of the genetically engineered T cell of any of embodiments 1-53 and 191, or the composition of any of embodiments 192-201, to a subject having the disease or disorder.

203. Use of the genetically engineered T cell any of embodiments 1-53 and 191, a plurality of the genetically engineered T cell of any of embodiments 1-53 and 191, or the composition of any of embodiments 192-201 for treating a disease or disorder.

204. Use of the genetically engineered T cell any of embodiments 1-53 and 191, a plurality of the genetically engineered T cell of any of embodiments 1-53 and 191, or the composition of any of embodiments 192-201 in the manufacture of a medicament for treating a disease or disorder.

205. The genetically engineered T cell any of embodiments 1-53 and 191, a plurality of the genetically engineered T cell of any of embodiments 1-53 and 191, or the composition of any of embodiments 192-201, for use in treating a disease or disorder.

206. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 202-205, wherein the disease or disorder is associated with HPV, optionally HPV 16.

207. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 202-206, wherein the disease or disorder is a cancer or a tumor, optionally a solid tumor.

208. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 207, wherein the tumor is associated with a cervical cancer, a uterine cancer, an anal cancer, a colorectal cancer, a vaginal cancer, a vulvar cancer, a penile cancer, a oropharyngeal cancers, a tonsil cancer, a pharyngeal cancers, a laryngeal cancer, an oral cancer, a skin cancer, a esophageal cancer, a head and neck cancer or a small cell lung cancer.

209. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 207 or 208, wherein the tumor is associated with a head and neck cancer, optionally a head and neck squamous cell carcinoma (HNSCC).

210. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 207 or 208, wherein the tumor is associated with a cervical cancer, optionally a cervical carcinoma.

211. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 202-210, wherein a dose of the genetically engineered T cells is administered to the subject.

212. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 211, wherein the dose of genetically engineered T cells comprises between at or about 3 x 10 ⁷ recombinant TCR-expressing T cells and at or about 3 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive.

213. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 211, wherein the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR-expressing T cells and at or about 1 x 10 ¹⁰ recombinant TCR-expressing T cells, inclusive.

214. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of embodiment 211, wherein the dose of genetically engineered T cells comprises between at or about 1 x 10 ⁸ recombinant TCR-expressing T cells and at or about 1 x 10 ⁹ recombinant TCR-expressing T cells, inclusive.

215. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213, wherein the dose of genetically engineered T cells comprises: at or about 1 x 10 ⁸ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR-expressing T cells; at or about 1 x 10 ⁹ recombinant TCR-expressing T cells; at or about 3 x 10 ⁸ recombinant TCR-expressing T cells; or at or about 1 x IO ¹⁰ recombinant TCR-expressing T cells.

216. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213 and 215, wherein the dose of genetically engineered T cells comprises at or about 1 x 10 ⁸ recombinant TCR- expressing T cells.

217. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213 and 215, wherein the dose of genetically engineered T cells comprises at or about 3 x 10 ⁸ recombinant TCR- expressing T cells.

218. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213 and 215, wherein the dose of genetically engineered T cells comprises at or about 1 x 10 ⁹ recombinant TCR- expressing T cells.

219. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213 and 215, wherein the dose of genetically engineered T cells comprises at or about 3 x 10 ⁹ recombinant TCR- expressing T cells.

220. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-213 and 215, wherein the dose of genetically engineered T cells comprises at or about 1 x 10 ¹⁰ recombinant TCR- expressing T cells.

221. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-215, wherein the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose; and/or the percentage of CD8+ T cells in the dose is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the dose. 222. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-215, wherein the dose of genetically engineered T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from at or about 1:3 to at or about 3:1, optionally at or about 1:1.

223. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-217, wherein interleukin- 2 (IL-2) or a variant thereof is further administered to the subject.

224. The method, use or the genetically engineered T cell, plurality of genetically engineered T cells or composition for use of any of embodiments 211-218, wherein the dose of genetically engineered T cells: is less sensitive to or resistant to immune suppression in the tumor microenvironment (TME), optionally immune suppression mediated by TGFp, when administered to the subject; results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to the subject; results in increased modified tumor control index (mTCI) when administered to the subject; exhibits a less terminally differentiated cell phenotype after exposure to an antigen that is recognized by the recombinant TCR; results in greater systemic expansion and/or longer persistence when administered to the subject; and/or results in a suppression of tumor growth, a reduction in tumor burden and/or an increase in survival of a subject, when administered to the subject at a dose that is lower than a dose of T cells engineered to express a comparator T cell receptor and/or a dose of T cells engineered that does not comprise the first genetic disruption.

IX. EXAMPLES

[0615] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Generation Engineered T Cells Expressing a Recombinant T Cell Receptor (TCR) by Targeted Knock-in (KI) at the T cell receptor alpha constant (TRAC) Locus and Knockout (KO) of Transforming Growth Factor Beta Receptor 2 (TGFBR2) [0616] Polynucleotides encoding exemplary recombinant T cell receptors (TCRs) were introduced into T cells with a genetic disruption at the endogenous gene locus that encode the T cell receptor alpha (TCRa) chain, by CRISPR/Cas9 mediated gene editing and targeted integration (targeted knock-in, KI) at the site of genetic disruption via homology-directed repair (HDR), with or without a genetic disruption to knockout (KO) the transforming growth factor beta receptor 2 (TGFBR2) locus.

A. Recombinant TCR Transgene Constructs

[0617] Exemplary template polynucleotides were generated for targeted integration by HDR of a transgene containing nucleic acid sequences encoding an exemplary recombinant TCR targeting the human papilloma virus (HPV) (TCR1; VP amino acid sequence set forth in SEQ ID NO: 1; CP amino acid sequence set forth in SEQ ID NO:2; Va amino acid sequence set forth in SEQ ID NO: 8; Ca amino acid sequence set forth in SEQ ID NO:9). The exemplary anti-HPV TCR comprises a fully human TCRaP sequence that is HLA-A*02:01-restricted, CD8 coreceptor- independent and targets the tumor-restricted HPV- 16 E7( 11-19) onco-peptide.

[0618] The general structure of the exemplary template polynucleotides were as follows: [5’ homology arm] -[transgene sequences] -[3’ homology arm]. The homology arms included approximately 600 bp of nucleic acid sequences homologous to sequences surrounding the target integration site in exon 1 of the human TCRa constant region (TRAC) gene (5’ homology arm sequence set forth in SEQ ID NO:56; 3’ homology arm sequence set forth in SEQ ID NO:57). The transgene included nucleic acid sequences encoding the TCRP and TCRa chains of the exemplary recombinant TCR (TCR1), separated by a 2A ribosome skip element.

B. Generation of Engineered T cells

[0619] Primary human CD4+ and CD8+ T cells were isolated by immunoaffinity-based selection from cryopreserved and thawed human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. CD8+ T cells were initially selected with CD8 selection beads, and CD4+ T cells were selected with CD4 selection beads. CD4+ and CD8+ cells were combined at a 1:1 ratio and stimulated by culturing with an anti-CD3/anti-CD28 Fab conjugated oligomeric reagent at 37°C in media.

[0620] For introducing a genetic disruption at the endogenous TCRa constant region (TRAC) locus by CRISPR/Cas9-mediated gene editing, the cells were electroporated with 1.7 to 2.5 pM ribonucleoprotein (RNP) complexes containing Streptococcus pyogenes Cas9 and TRAC-targeting guide RNA (gRNA) with targeting domain sequences GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:28; targeting within exon 1 of the endogenous TRAC gene). For introducing a genetic disruption at the growth factor beta receptor 2 (TGFBR2) locus to knock out TGFBR2, cells were co-electroporated with the RNP containing TRAC-targeting gRNA as described above and 2 to 2.5 pM RNP complexes containing TGFBR2-targeting gRNA with targeting domain sequences GUGGAUGACCUGGCUAACAG (SEQ ID NO:58; targeting within exon 4 of the endogenous TGFBR2 gene). For targeted integration (KI) of transgene sequences at the endogenous TRAC locus, cells were transduced by addition of adeno-associated virus (AAV) preparation containing a homology-directed repair (HDR) template polynucleotide encoding an exemplary recombinant TCR (TCR1) as described in Example l.A above, to generate cells engineered to express the recombinant TCR by knock- in at the TRAC locus and knockout of the TGFBR2 locus (engineered cells designated TCR1 TRAC KI TGFBR2 KO). Cells engineered to express the recombinant TCR by knock-in at the TRAC locus without TGFBR2 KO (designated TCR1 TRAC KI TGFBR2 WT), or cells with a genetic disruption at the TRAC locus (TRAC KO) and/or TGFBR2 locus (TGFBR2 KO) without the recombinant TCR, were assessed as controls.

[0621] Following electroporation, cells were incubated for recovery and expansion in serum- free media.

C. Knock out Efficiency and Expression of TCRs

[0622] Expression of the engineered cells in each of the groups was assessed by flow cytometry for staining with an anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, and an anti-Vbeta2 antibody specific for the exemplary recombinant TCR.

[0623] Results are shown in FIG. 1A and Table El below. Electroporation with RNPs complexed with gRNAs targeting TGFBR2 resulted in efficient knock-out of TGFBR2 (see Table El). TGFBR2 KO was evaluated molecularly by amplicon sequencing of edited cells versus unedited controls. Electroporation with RNPs complexed with gRNAs targeting TRAC resulted in high level and efficient knock-out of the endogenous TCR in T cells, as evidenced by flow cytometry staining with the anti-CD3 antibody for the endogenous TCR complex (FIG. 1A and Table El). Further, TCR1 TGFBR2 KO engineered cells showed high level expression of the recombinant TCR when targeted to the TRAC locus by HDR-mediated knock-in, which was comparable to the expression of the TCR1 in TCR1 TGFBR2 WT cells (left panels of FIG. 1A and Table El). The results also demonstrated a similar CD4+:CD8+ cell ratios in the engineered cell compositions, indicating that the methods of engineering and editing the cells did not impact the resulting CD4 to CD8 ratio. The results support the generation of T cells with high efficiency expression of an exemplary recombinant TCR when integrated at the endogenous TRAC locus and containing a TGFBR2 KO.

Table El. Knock-out and Knock-in Efficiency for TRAC or TGFBR2

J assessed by flow cytometry; assessed by RNP re-cutting

D. Expression of recombinant TCR with TRAC KO

[0624] Expression of the exemplary TCR in T cells knocked-out for the endogenous TCR was compared in cells introduced with the recombinant TCR by HDR-mediated knock-in (TCR1 TRAC KI) or by non-targeted lentiviral transduction (TCR1 TRAC KO). Specifically, T cells knocked out for the endogenous TCRa constant region (TRAC) locus by methods described above were transduced with a lentiviral construct encoding the exemplary recombinant TCR1 (TCR1 TRAC KO) or the AAV KI construct as described in Example l.A (TCR1 TRAC KI), or were mock-transduced (mock) using the respective transduction method.

[0625] Cells were assessed by flow cytometry by staining with an anti-CD3 antibody, anti- CD4 antibody, anti-CD8 antibody, an anti-Vbeta2 antibody that recognizes TCRs with the Vbeta variable domain, and with a peptide-MHC dextramer complexed with the HPV-16 E7 peptide (multivalent HLA-A*02:01/HPV E7(l l-19)).

[0626] As shown in FIG. IB, staining with anti-Vbeta2 indicated high efficiency knock-out of the endogenous TCR in the engineered T cells, as indicated by only 6% or 10% expression in mock-transduced CD4+ and CD8+ T cells, respectively. In T cells transduced with the recombinant TCR1, the frequency of cells expressing a TCR increased, particularly cells double positive for staining with anti-Vbeta2 and the peptide-MHC dextramer, demonstrating the expression of the exemplary recombinant TCR1 in the engineered T cells. The expression of TCR1 was substantially improved in cells engineered by TRAC KI than in the cells engineered by non-targeted lentiviral transduction into TRAC KO T cells, both in terms of the frequency of T cells expressing the recombinant TCR and the higher and more homogenous levels of TCR1 expression. The results support increased and more homogeneous expression of an exemplary recombinant TCR when integrated at the endogenous TRAC locus.

Example 2: In vitro Functional Assessment of Anti-tumor Activity [0627] The response to tumor antigen and anti-tumor activity of engineered T cells expressing an exemplary TCR by knock-in at the endogenous TCRa constant region (TRAC) locus and with knockout of the TGFBR2 locus was assessed by a spheroid killing assay after prolonged stimulation. The engineered cells also were assessed for resistance to inhibition of TGFP signaling.

[0628] The engineered T cells generated as described in Example l.B (TCR1 TRAC KI TGFBR2 KO cells and TCR1 TRAC KI TGFBR2 WT cells), were subjected to a 10-day stimulation by incubation with human HPV+ SCC-152 head and neck squamous cell carcinoma (HNSCC) cell line tumor spheroids, or CasKi human cervical cancer cell line tumor spheroids at an effector: target (E:T) ratio of 1:2, 1:10 or 1:20. The E:T ratio of 1:20 was a sub-optimal ratio (e.g., low number of effector cells compared to the target) that permits further distinction between differences in the effector activity of various groups. Cells were re-stimulated with tumor spheroids again starting day 10 at 1:20 E:T ratio. The tumor spheroids were labeled with a red fluorescent dye to permit monitoring of tumor cell lysis (using the IncuCyte® Live Cell Analysis System, Essen Bioscience). Tumor spheroid size was monitored, by fluorescence microscopy, to determine anti-tumor activity until day 20, as an extended antigen stimulation condition to reflect chronic stimulation. The TCR1 TRAC KI TGFBR2 WT cells and the TCR1 TRAC KI TGFBR2 KO cells exhibited more than 95% TRAC KO, more than 80% TGFBR2 KO and more than 75% HPV TCR KI, as assessed by flow cytometry and molecular characterization.

[0629] Target cells without engineered T cells (target only) were used as controls. At day 3, 5, 9, 19, and 20 of stimulation with tumor spheroids, cells cultured at the E:T ratio of 1:20 were harvested and assessed by flow cytometry for phenotype of the engineered cells by staining with the following antibodies: an anti-CDlOl antibody and an anti-CD38 antibody (exemplary markers for terminally differentiated cells and/or cells resistant to immuno-reprograming), an anti-CD25 antibody, an anti-CD27 antibody, an anti-PD-1 antibody, and an anti-TIM-3 antibody (exemplary markers for differentiated phenotypes). At day 3, 10 and 20, the level of cytokines interferon-gamma (IFN-y), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-a) were assessed using a multiplex cytokine assay kit.

A. Tumor Spheroid Killing

[0630] Tumor spheroid killing was assessed by monitoring the changes in the tumor spheroid size, with smaller tumor spheroid size representing greater anti-tumor activity, after restimulation with a spheroid between days 10 and 20. As shown in FIG. 2A, the exemplary anti- HPV TCR-expressing cells with a knockout of the TGFBR2 gene (TCR1 TRAC KI TGFBR2 KO) exhibited the greatest reduction in spheroid size, compared to cells expressing the same exemplary anti-HPV TCR without the knockout of TGFBR2 gene (TCR1 TRAC KI TGFBR2 WT).

[0631] As shown in FIG. 2B in photographs of representative tumor cell spheroids, the reduction in spheroid size (as indicated by reduced red fluorescence) at day 20 was greater in TCR1 TRAC KI TGFBR2 KO cells compared to TCR1 TRAC KI TGFBR2 WT cells. The results showed improved cytolytic function and complete tumor spheroid clearance by TCR1 TRAC KI TGFBR2 KO cells even at a suboptimal effector to target ratio of 1:20.

[0632] In a similar study, the difference between cytotoxic activity of TCR1 TRAC KI TGFBR2 KO cells and TCR1 TRAC KI TGFBR2 WT cells at a high (e.g., optimal) and low (e.g., sub-optimal) E:T ratio, in a tumor spheroid assay. The TCR1 TRAC KI TGFBR2 KO cells and TCR1 TRAC KI TGFBR2 WT cells were stimulated with HNSCC HPV+ SCC152 tumor spheroids at E:T ratios of 1:2 (optimal E:T) or 1:20 (sub-optimal E:T) for 10 days, followed by restimulation with tumor spheroids on Day 10, and monitoring of tumor spheroid size until Day 20. As shown in FIG. 2C (optimal E:T), TCR1 TRAC KI TGFBR2 KO cells exhibited similar cytolytic function compared to TCR1 TRAC KI TGFBR2 WT cells, at an optimal E:T ratio of 1:2. However, as shown in FIG. 2D (sub-optimal E:T), TCR1 TRAC KI TGFBR2 KO cells exhibited substantially improved cytolytic function compared to TCR1 TRAC KI TGFBR2 WT cells, at a sub-optimal E:T ratio of 1:20, which permits further distinction between differences in the effector activity of various groups.

[0633] The results were consistent with an observation that engineered T cells expressing an exemplary anti-HPV TCR by KI at the TRAC locus and also containing a TGFBR2 KO demonstrated improved anti-tumor activity against tumor spheroids compared to T cells expressing the same TCR by KI at the TRAC locus but without a TGFBR2 KO. Similar results were observed using T cells obtained from different donors or stimulated using a different tumor target cell line.

B. Cell Phenotypes

[0634] As shown in FIGS. 3A, extended stimulation of the T cells at an effector to target ratio of 1:20 also resulted in changes in the cell phenotype of T cells engineered with a recombinant anti-HPV TCR by KI at the TRAC locus and with TGFBR2 KO (TCR1 TRAC KI TGFBR2 KO cells) compared to T cells expressing the same TCR but without a TGFBR2 KO (TCR1 TRAC KI TGFBR2 WT cells), as evidenced by staining for CD38 and CD101. Following tumor antigen encounter, T cells undergo chromatin remodeling and initially remain reprogrammable with low expression levels of CD38 and CD101, whereas as cells that differentiate into a fixed chromatin state can be resistant to reprogramming and express high levels of CD38 and CD101. Thus, CD38+CD1O1+ cells provide a surrogate to identify terminally differentiated cells that may be resistant to immune-reprogramming cells. As shown in FIG. 3A and FIG. 3B, the results demonstrated that after extended antigen stimulation at days 3, 5, 9, and 19, TCR1 TRAC KI TGFBR2 KO cells had a lower percentage of CD101+ CD38+ T cells compared to TCR1 TRAC KI TGFBR2 WT cells. The results were consistent with an observation that the TCR1 TRAC KI TGFBR2 KO cells showed lower levels of terminal differentiation and exhaustion compared to the TCR1 TRAC KI TGFBR2 WT cells. Similar results were observed using T cells obtained from different donors or stimulated using a different tumor target cell line.

[0635] As shown in FIG. 3C, assessment of other immunosuppressive or activation markers after extended antigen stimulation also demonstrated that TCR1 TRAC KI TGFBR2 KO cells exhibited lower expression of CD25, CD27, PD-1 and TIM-3 compared to cells expressing the same engineered anti-HPV TCR without the knockout of TGFBR2, as evidenced by lower geometric mean fluorescence intensity (gFMI) by flow cytometry staining. These results further demonstrate that TCR1 TRAC KO TGFBR2 KO cells exhibited a less differentiated phenotype after extended antigen stimulation. Without wishing to be bound by theory, the results support that KO of TGFBR2 in the engineered T cells may de-couple T cell expansion and onset of exhaustion, particularly in solid tumor microenvironments exposed to the immunosuppressive cytokine TGFp.

C. In vitro cell expansion

[0636] In vitro cell expansion of the engineered T cells expressing the recombinant anti- HPV TCR was assessed in the spheroid assay described above by determining the number of live cells and the fold expansion after the first stimulation and the re-stimulation with tumor spheroids.

[0637] FIG. 4A shows representative images of replicates of tumor cell spheroids that were incubated with TCR1 TRAC KI TGFBR2 KO cells and TCR1 TRAC KI TGFBR2 WT cells, in a tumor spheroid re-stimulation experiment, generally as described in Example 2. A, at a sub- optimal E:T ratio (1:20), at day 20. As shown, the TCR1 TRAC KI TGFBR2 KO cells led to substantial reduction of tumor spheroids, whereas the labeled tumor cell spheroids were still visible when incubated with TCR1 TRAC KI TGFBR2 WT cells. [0638] FIGS. 4B-4C show the number of live cells and fold cell expansion of TCR1 TRAC KI TGFBR2 KO and TCR1 TRAC KI TGFBR2 WT cells, after the initial stimulation at day 10 (FIG. 4B) and after re- stimulation at day 20 (FIG. 4C), at an E:T ratio of 1:2, 1:10 or 1:20. As shown, the fold expansion was higher in TCR1 TRAC KI TGFBR2 KO cells at E:T ratios 1:10 or 1:20, compared to TCR1 TRAC KI TGFBR2 WT cells, after the first stimulation. After the re- stimulation to reflect chronic stimulation, TCR1 TRAC KI TGFBR2 KO cells exhibited higher fold expansion at compared to TCR1 TRAC KI TGFBR2 WT cells, at E:T ratios 1:10 or 1:20. TCR1 TRAC KI TGFBR2 KO cells increased approximately 20-fold and 40-fold at 1:10 and 1:20 E:T ratios, respectively. In contrast, less than 10-fold cell expansion was observed for TCR1 TRAC KI TGFBR2 WT cells at Day 10. Similarly, greater cell expansion was observed at Day 20 for TCR1 TRAC KI TGFBR2 KO cells in response to chronic stimulation conditions (e.g., after re- stimulation) at a sub-optimal E:T ratio. An approximately 10-fold expansion of TCR1 TRAC KI TGFBR2 KO cells was observed, compared to less than 2-fold for TCR1 TRAC KI TGFBR2 WT cells at the E:T ratio of 1:20. These results indicate that TCR1 TRAC KI TGFBR2 KO cells show greater expansion than TCR1 TRAC KI TGFBR2 WT cells in response to chronic stimulation conditions.

D. Cytokine Production

[0639] FIG. 4D shows the level of cytokines interferon-gamma (IFN-y), interleukin-2 (IL- 2), and tumor necrosis factor-alpha (TNF-a) produced in the tumor spheroid co-culture, at days 3, 10 and 20, at E:T ratios of 1:2, 1:10 or 1:20. As shown, the TCR1 TRAC KI TGFBR2 KO cells generally produced more cytokines in response to stimulation with the tumor spheroid coculture, compared to TCR1 TRAC KI TGFBR2 WT cells. In general, the difference in cytokine production was more markedly observed at the sub-optimal E:T ratio (e.g., 1:20), and often at the later time point (e.g., 20 days).

E. Conclusion

[0640] The results supported that under chronic antigen stimulation and in the presence of high levels of the immunosuppressive cytokine TGFp, at an optimal effector-to-target (E:T) ratio, an exemplary HPV 16-targeting recombinant TCR engineered T cells and T cells expressing the same recombinant TCR but also comprising a KO of the TGFBR2 locus, demonstrated robust and comparable cytotoxic functions in vitro. However, when tested at a suboptimal E:T ratio, HPV TCR-expressing TGFBR2 KO cells demonstrated superior expansion (for example, a 5-fold higher expansion), cytotoxicity and cytokine production, and improved phenotypes, for example, as demonstrated by a reduction in phenotypes indicative of exhaustion. The results were consistent with an observation that TGFP-armoring (e.g., by reducing the expression of TGFBR2 by a genetic disruption) may decouple T cell expansion and the onset of exhaustion. The results support that inhibiting TGFP-mediated immune suppression, for example by genetic disruption at the TGFBR2 gene in an exemplary HPV 16-targeting recombinant TCR engineered T cells, achieves improved activity and function of the engineered T cells. Similar results were observed using engineered cells from another donor as well as using the CasKi tumor model. The improvements in anti-tumor activity, phenotype and expansion were often observed more apparently at a low or sub-optimal E:T ratio.

Example 3: In vivo Functional Assessment of Anti-tumor Activity

[0641] The anti-tumor activity, in vivo persistence and expansion, and phenotype of the exemplary HPV 16-targeting recombinant TCR-expressing cells, with or without knockout (KO) of the transforming growth factor beta receptor 2 (TGFBR2) locus, were assessed using two different HPV-positive tumor-bearing mouse xenograft models: SCC-152 human head and neck squamous cell carcinoma (HNSCC) and CasKi human cervical cancer.

A. Assessment of in vivo anti-tumor effects in a mouse xenograft model of head and neck squamous cell carcinoma (HNSCC)

[0642] A mouse tumor model of head and neck squamous cell carcinoma (HNSCC) was generated by subcutaneous injection of 5 x 10 ⁶ squamous cell carcinoma cell line UPCESCC- 152 (ATCC® CRL-3240™) cells, into NOD.Cg-Prkdc ^scid h2rg ^tmlwjl /SzJ (NSG) mice. The UPCLSCC-152 cell line originates from a recurrent squamous cell carcinoma of the hypopharynx, and are positive for HLA-A*02:01 and the HPV 16 E7 oncoprotein. Approximately 3 weeks after injection, at which time the tumor-bearing mice exhibited a mean tumor volume of about 150 mm ³ , the mice were staged and divided into groups (n=8 mice/group). At staging, TGFP levels were measured in the tumor sample. As shown in FIG. 5A, the SCC-152 tumor xenograft model expressed high levels of TGFP in the tumor, which can impact the function of the engineered T cells expressing a recombinant TCR.

[0643] Primary human CD4+ and primary human CD8+ T cells obtained from two different human donors were engineered to express the exemplary anti-HPV 16 TCR1, substantially as described in Example 1A above. Mice were administered 1 x 10 ⁶ (higher dose) or 3.33 x 10 ⁵ (lower, sub-optimal dose) TCR-expressing T cells for the TCR1 TRAC KI TGFBR2 KO and TCR1 TRAC KI TGFBR2 WT groups, or 1 x 10 ⁶ TRAC KO, TGFBR2 KO cells as a control, at Day 0.

[0644] Mean tumor volume, body weight, and survival were assessed twice a week for up to approximately 60 days after administration of the engineered T cells, and the number of TCR- expressing CD4+ T cells and CD8+ T cells were assessed after administration of the engineered T cells. Surface expression of CD 103 in tumor infiltrating lymphocytes (TIL) that express the recombinant TCR was also assessed after administration of the engineered cells. CD 103 is a pharmacodynamics marker downstream of the TGFP activation pathway, indicative of TGFP signaling and immunosuppression. For example, in engineered T cells that are not knocked-out for TGFBR2, the level of CD103-expressig TIL were elevated in the SCC-152 model, with increased expression on TCR1 TRAC KI TGFBR2 WT cells at day 14 compared to day 7 in this model (FIG. 5B). The elevated levels of TGFP mimic those of human tumors, and can impact the function of anti-HPV TCR-expressing T cells, as shown by elevated CD 103 staining (surrogate marker downstream of the TGFP activation pathway) in tumor infiltrating lymphocytes (TIL). The number of live circulating CD45+/CD3+/eTCR+ T cells and CD1O3+/CD3+ T cells in in the mice was assessed by multicolor flow cytometry on days 6, 13, 20, and 27 or day 13, respectively, after administration of the engineered T cells.

[0645] For individual tumor growth curves, the modified Tumor Control Index (mTCI) score, which is an aggregate scoring method for determining the ability of therapeutic interventions to control tumor growth based on a numerical score of 5 equally weighted components: inhibition (normalized over time interval), stabilization (normalized to tumor volume), suppression, dynamic control, and extremal inhibition, was determined, using R Shiny app (version 3.6.0). Statistical analysis of mTCI calculations was performed using a nonparametric Kruskal-Wallis test with a false discovery rate (FDR) correction for multiple comparisons within each dose level.

1. Anti-tumor Activity

[0646] Administration of TCR1 TRAC KI TGFBR2 KO cells exhibited higher anti-tumor activity in this model compared to cells expressing TCR1 TRAC KI TGFBR2 WT, as evidenced by a substantially complete elimination of tumor volume starting around day 28, through the end of the study. The improved in vivo anti-tumor activity was more apparent at a lower dose (3.33 x 10 ⁵ ; FIG. 6A) compared to a higher dose (l x 10 ⁶ ; FIG. 6B). Results for individual mice is shown in FIG. 6C.

[0647] The modified Tumor Control Index (mTCI) score of tumor growth in mice administered TCR1 TRAC KI TGFBR2 KO cells was substantially higher compared to in mice administered the TCR1 TRAC KI TGFBR2 WT cells at the lower dose (FIG. 6D, dose of 3.33 x 10 ⁵ cells); the improvement was to a lower degree at the higher dose (FIG. 6E, dose of 1 x 10 ⁶ cells). As shown in FIG. 6F, administration of TCR1 TRAC KI TGFBR2 KO cells exhibited improved tumor-free survival compared to mice administered TCR1 TRAC KI TGFBR2 WT cells or TRAC KO or TGFBR2 KO control T cells. At the last time point assessed in this study depicted in FIG. 6F, 100% of the mice survived following administration of TCR1 TRAC KI TGFBR2 KO cells. The results support substantial inhibition of tumor growth with TCR1 TRAC KI TGFBR2 KO cells compared to TCR1 TRAC KI TGFBR2 WT cells treatment (p <0.001) at a sub-optimal dose of 3.3xl0 ⁵ cells, in SCC-152 HNSCC HPV+ tumor xenograft mice. Also, mice that received TCR1 TRAC KI TGFBR2 KO cells at a sub-optimal 3.3xl0 ⁵ dose exhibited a substantial survival advantage over mice receiving TCR1 TRAC KI TGFBR2 WT cells (p <0.05 at 3.3xl0 ⁵ , log-rank [Mantel-Cox] test followed by Benjamini-Hochberg post-test correction for multiple comparisons).

2. Cell Expansion

[0648] Analysis of circulating T cells at days 6, 12, 20 and 27 after their administration to tumor-bearing mice in this model demonstrated that TCR1 TRAC KI TGFBR2 KO cells remained circulating in blood longer (through at least day 27) compared to TCR1 TRAC KI TGFBR2 WT cells or TRAC KO or TGFBR2 KO control T cells, as shown for both CD4+ T cells (FIG. 7A) and CD8+ T cells (FIG. 7B). The results demonstrate that cells expressing an HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibited improved systemic expansion in peripheral blood. In addition, weight loss, typically associated with graft-versus- host disease (GvHD) was not observed.

3. TGFp Signaling

[0649] At day 13, cells were harvested and assessed by flow cytometry by staining with an anti-CD103 antibody and anti-CD3 antibody. As shown in FIG. 8A (dose of 3.33 x 10 ⁵ cells) and FIG. 8B (dose of 1 x 10 ⁶ cells), TCR1 TRAC KI TGFBR2 KO cells exhibited reduced TGFP-mediated signaling, as evidenced by a near complete absence in CD103-expressing cells among these cells compared to TCR1 TRAC KI TGFBR2 WT cells. The results are consistent with the observation that TGFBR2 KO in cells efficiently blocked TGFP signaling in this model.

[0650] Taken together, the results were consistent with an observation that inhibition of TGFP-mediated immune suppression, by a knockout of the TGFBR2 gene in HPV 16-targeted TCR engineered T cells results in improved anti-tumor activity and survival of mice administered with such cells. The results are consistent with an observation that T cells engineered to express a HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibit substantially improved and prolonged anti-tumor activity in vivo. B. Assessment of in vivo anti-tumor effects in a mouse xenograft model of cervical cancer

[0651] A different mouse tumor model, of cervical cancer, was generated by subcutaneous injection of 5 x 10 ⁶ CasKi human cervical cancer cell line into NOD.Cg-Prkdc ^scld I12rg ^{tml wjl} /SzJ (NSG) mice. The CasKi cells originated from a metastasis in the small bowel mesentery and are positive for HPV. Approximately 18 days after injection, at which time the tumor-bearing mice exhibited a mean tumor volume of about 100 mm ³ , the mice were staged and divided into groups. Similar to the SCC152 model described above, the CasKi tumor xenograft model exhibited features of an immunosuppressive tumor microenvironment. This was evidenced by expression of TGFP in the tumor although at a level less than the SCC-152 tumor model (FIG. 5A, and elevated levels of CD 103 expression on TCR1 TRAC KI TGFBR2 WT cells at day 14 compared to day 7 after administration of the engineered cells (FIG. 5B).

[0652] The mice of each group were intravenously administered 10 x 10 ⁶ TCR-expressing cells for the TCR1 TRAC KI TGFBR2 KO and TCR1 TRAC KI TGFBR2 WT groups, or 10 x 10 ⁶ for TRAC KO, control KO cells. In this study, the mean tumor volume was assessed twice a week for up to 70 days after administration of the engineered cells, and modified Tumor Control Index (mTCI), body weight, survival, number of live circulating TCR-expressing cells, CD45+/CD3+/eTCR+ T cells and CD1O3+/CD3+ T cells were assessed after administration of the engineered cells, generally as described above.

1. Anti-tumor Activity

[0653] Administration of TCR1 TRAC KI TGFBR2 KO cells exhibited higher anti-tumor activity in this model compared to cells expressing TCR1 TRAC KI TGFBR2 WT cells, as evidenced by a substantially complete elimination of tumor volume starting around day 35, through the end of the study, as shown in FIG. 9A (mean tumor volume) and in FIG. 9B (tumor volume of individual mice per group).

[0654] The modified Tumor Control Index (mTCI) score of tumor growth in mice administered TCR1 TRAC KI TGFBR2 KO cells was substantially higher compared to mice administered TCR1 TRAC KI TGFBR2 WT cells (FIG. 9C), demonstrating a greater ability of TCR1 TRAC KI TGFBR2 KO cells to control tumor growth. As shown in FIG. 9D, administration of TCR1 TRAC KI TGFBR2 KO cells resulted in substantially improved tumor- free survival compared to mice administered TCR1 TRAC KI TGFBR2 WT cells or TRAC KO, TGFBR2 KO control cells. At the last time point assessed in this study depicted in FIG. 9D, 100% of the mice survived following administration of TCR1 TRAC KI TGFBR2 KO cells.

[0655] The results further show that T cells expressing an HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibit substantial anti-tumor activity in vivo and prolonged survival, also in a model of cervical cancer. The anti-tumor activity was substantially improved compared to the cells expressing the same TCR without TGFBR2 knockout or control groups (p <0.001).

2. Cell Expansion

[0656] As shown in FIGS. 10A-10B, results showed that TCR1 TRAC KI TGFBR2 KO cells remained circulating in blood longer (through at least day 28) compared to TCR1 TRAC KI TGFBR2 WT cells or TRAC KO or TGFBR2 KO control T cells, as shown for both CD4+ T cells (FIG. 10A) and CD8+ T cells (FIG. 10B). Similar to the results in the SCC152 model, the results demonstrate that T cells expressing a HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibited improved systemic expansion in peripheral blood.

3. TGFp Signaling

[0657] Similar to the SCC152 model, TCR1 TRAC KI TGFBR2 KO cells exhibited reduced TGFP-mediated signaling, as evidenced by a near complete reduction of CD 103 expression on the TCR-expressing CD3+ T cells in this model at day 14 after administration of the T cells (FIG. 10C). The results are consistent with the observation that administration of TGFBR2 KO cells in this model efficiently blocks TGFP signaling.

C. Conclusion

[0658] The results of the in vivo assessment, using two different tumor models and T cells obtained from different donors, support the improved anti-tumor activity of T cells engineered with an HPV 16-targeted TCR and with a knockout (KO) of the TGFBR2 locus, compared to T cells expressing the same recombinant TCR without a TGFBR2 KO. The results show blocking of TGFP signaling, improved anti-tumor activity as evidenced by reduction of tumor volume and increased mTCI score, and prolonged survival of tumor xenograft mice, without weight loss (indicative of GvHD). The results also show improved systemic expansion of the administered cells with a TGFBR2 KO. Such differences were more apparent when the cells were administered at a lower dose, supporting robust anti-tumor activity and expansion of the engineered cells even at a sub-optimal dose.

[0659] The results support effective anti-tumor activity of the cells expressing the exemplary TCR with a TGFBR2 KO, for example to counter TGFP-mediated immunosuppression, even when administered at a lower dose, and in some aspects, the administration of a lower dose of engineered T cells. Administration of lower doses is supported by the results described herein, which demonstrate that the TCR1 TRAC KI TGFBR2 KO cells show improved anti-tumor effects, pharmacodynamics characteristics (e.g., improved expansion) and phenotypic characteristics (e.g., reduced exhaustion), even when administered at a lower dose. Administration of a lower dose can also provide additional advantages of improved safety profiles, and reducing or minimizing potential adverse effects and the time and cost necessary to produce the dose of cells.

Example 4: Assessment of in vivo anti-tumor effects in a mouse xenograft model of head and neck squamous cell carcinoma (HNSCC) expressing high levels of TGFQ

[0660] A mouse tumor model of head and neck squamous cell carcinoma (HNSCC) expressing higher levels of TGFP than the UPCI:SCC-152 model was assessed to determine whether knock out (KO) of TGFBR2 in the administered exemplary HPV 16-targeting recombinant TCR-expressing T cells, would confer greater anti-tumor benefit compared to the TCR-expressing T cells without KO of TGFBR2 (TGFBR2 WT).

[0661] The mouse tumor model was generated by subcutaneous injection of 5 x 10 ⁵ squamous cell carcinoma cell line UM-SCC-104 cells, into NOD.Cg-Prkdc ^scld I12rg ^{tml wjl} /SzJ (NSG) mice. As shown in FIG. 12, UM-SCC-104 cells express substantially higher levels of TGFP than UPCLSCC-152 and Caski cells. Approximately 13 days after injection, at which time the tumor-bearing mice exhibited a mean tumor volume of about 100 mm ³ , the mice were staged and divided into groups (n=8 mice/group).

[0662] Primary human CD4+ and primary human CD8+ T cells obtained from two different human donors were engineered to express the exemplary anti-HPV 16 TCR1, substantially as described in Example 1A above. Mice were administered 6 x 10 ⁶ (high dose), 2 x 10 ⁶ (medium dose) or 6 x 10 ⁵ (low dose) TCR-expressing T cells for the TCR1 TRAC KI TGFBR2 KO and TCR1 TRAC KI TGFBR2 WT groups, or 6 x 10 ⁶ double knockout (TRAC KO, TGFBR2 KO) cells (Mock) as a control, at Day 0.

[0663] Mean tumor volume, body weight, and survival were assessed twice a week for up to approximately 65 days after administration of the engineered T cells, and the number of TCR expressing CD4+ T cells and CD8+ T cells were assessed after administration of the engineered T cells.

A. Anti-tumor Activity

[0664] Administration of TCR1 TRAC KI TGFBR2 KO cells resulted in anti-tumor activity at each dose level, with tumor control and some survival benefit at the low dose, and complete elimination of tumor volume with 100% survival at the medium and high doses. By contrast, administration of TCR1 TRAC KI TGFBR2 WT cells at each dose level did not result in statistically significant tumor volume reduction relative to the mock control and/or tumor alone groups. Mean tumor volume is shown in FIG. 13A (low dose), FIG. 13B (medium dose), and FIG. 13C (high dose). Results for individual mice is shown in FIG. 14A (low dose), FIG. 14B (medium dose), and FIG. 14C (high dose).

[0665] The modified Tumor Control Index (mTCI) score of tumor growth in mice administered TCR1 TRAC KI TGFBR2 KO cells was substantially higher compared to in mice administered the TCR1 TRAC KI TGFBR2 WT cells at each dose level (FIG. 15A (low dose), FIG. 15B (medium dose), and FIG. 15C (high dose)). As shown in FIG. 16A (low dose), FIG. 16B (medium dose), and FIG. 16C (high dose), administration of TCR1 TRAC KI TGFBR2 KO cells exhibited improved tumor-free survival compared to mice administered TCR1 TRAC KI TGFBR2 WT cells or Mock (TRAC KO, TGFBR2 KO) control T cells.

B. Cell Expansion

[0666] Analysis of circulating T cells at days 8, 15, 22, and 29 after their administration to tumor-bearing mice in this model demonstrated that TCR1 TRAC KI TGFBR2 KO cells remained circulating in blood longer (through at least day 29) compared to TCR1 TRAC KI TGFBR2 WT cells or Mock (TRAC KO, TGFBR2 KO) control T cells. Cells expressing an HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibited improved systemic expansion in peripheral blood. In addition, weight loss was not observed in the mice.

C. TGFp Signaling

[0667] At day 8, cells from the high dose group were harvested and assessed by flow cytometry by staining with an anti-CD103 antibody and anti-CD3 antibody. As shown in FIG. 17, TCR1 TRAC KI TGFBR2 KO cells exhibited reduced TGFP-mediated signaling, as evidenced by a near complete absence in CD103-expressing cells among these cells compared to TCR1 TRAC KI TGFBR2 WT cells.

[0668] Taken together, the results were consistent with an observation that inhibition of TGFP-mediated immune suppression, by a knockout of the TGFBR2 gene in HPV 16-targeted TCR engineered T cells results in improved anti-tumor activity and survival of mice administered with such cells. The results are consistent with an observation that T cells engineered to express a HPV 16-targeted TCR with a knockout of the TGFBR2 gene exhibit substantially improved and prolonged anti-tumor activity in vivo, including against tumor cells that express high level of TGFp.

Example 5: Administration of Engineered T Cells Expressing a Recombinant TCR by Targeted KI at the TRAC Locus and KO of TGFBR2 to Sub jects with Human Papilloma Virus (HPV) Positive Tumors

[0669] Compositions of TCR1 TRAC KI TGFBR2 KO cells, comprising T cells expressing an exemplary anti-human papilloma virus (HPV) TCR engineered by knock-in at the endogenous TCRa constant region (TRAC) locus and knockout of the TGFBR2 locus to inhibit TGFP signaling generated generally as described in Example 1, are administered to subjects with advanced, refractory, HPV- 16 positive solid cancers and an Human Leukocyte Antigen (HLA)-A*02:01 allele. Subjects with locally advanced or metastatic HPV- 16 positive solid tumors who have progressed on standard anticancer therapy, with medical comorbidities, intolerability, or for whom no other approved conventional therapy exists, are considered for administration.

[0670] At the initial dose-escalation stage, subjects receive 1 x 10 ⁸ , 3 x 10 ⁸ , 1 x 10 ⁹ , 3 x 10 ⁹ , or 1 x 10 ¹⁰ TCR1 TRAC KI TGFBR2 KO cells. Such doses in some cases are lower than the number of cells administered for T cells engineered with a different anti-HPV TCR, for example, of 1 x 10 ¹⁰ or 1 x 10 ¹¹ cells.

[0671] At the dose-expansion stage, subjects are divided into two groups: (i) subjects with advanced HPV-16 positive head and neck squamous cell carcinoma (HNSCC), and (i) subjects with other advanced HPV-16 positive solid tumors. Subjects are treated with maximum tolerated dose identified at the dose-escalation stage.

[0672] In some aspects, the safety and tolerability of increasing dose levels of the engineered cells is evaluated. In some aspects, response to treatment is assessed based on tumor burden measurements, pharmacokinetics, pharmacodynamics, and tumor infiltration of the engineered cells, physical examination findings, and various laboratory assessments.

[0673] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Sequences

gtgtgactttgggctttccctgcgtctggaccctactctgtctgtggatgacctggct aacagtgggcaggtgggaactgcaagatacatggctccagaagtcctagaatccagga tgaatttggagaatgttgagtccttcaagcagaccgatgtctactccatggctctggt gctctgggaaatgacatctcgctgtaatgcagtgggagaagtaaaagattatgagcct ccatttggttccaaggtgcgggagcacccctgtgtcgaaagcatgaaggacaacgtgt tgagagatcgagggcgaccagaaattcccagcttctggctcaaccaccagggcatcca gatggtgtgtgagacgttgactgagtgctgggaccacgacccagaggcccgtctcaca gcccagtgtgtggcagaacgcttcagtgagctggagcatctggacaggctctcgggga ggagctgctcggaggagaagattcctgaagacggctccctaaacactaccaaatagct cttctggggcaggctgggccatgtccaaagaggctgcccctctcaccaaagaacagag gcagcaggaagctgcccctgaactgatgcttcctggaaaaccaagggggtcactcccc tccctgtaagctgtggggataagcagaaacaacagcagcagggagtgggtgacataga gcattctatgcctttgacattgtcataggataagctgtgttagcacttcctcaggaaa tgagattgatttttacaatagccaataacatttgcactttattaatgcctgtatataa atatgaatagctatgttttatatatatatatatatatctatatatgtctatagctcta tat at at agecat acct tgaaaagagacaaggaaaaacatcaaat at tcccaggaaat tggttttattggagaactccagaaccaagcagagaaggaagggacccatgacagcatt agcatttgacaatcacacatgcagtggttctctgactgtaaaacagtgaactttgcat gaggaaagaggctccatgtctcacagccagctatgaccacattgcacttgcttttgca aaataatcattccctgcctagcacttctcttctggccatggaactaagtacagtggca ctgtttgaggaccagtgttcccggggttcctgtgtgcccttatttctcctggactttt catttaagctccaagccccaaatctggggggctagtttagaaactctccctcaaccta gtttagaaactctaccccatctttaataccttgaatgttttgaaccccactttttacc ttcatgggttgcagaaaaatcagaacagatgtccccatccatgcgattgccccaccat ctactaatgaaaaattgttctttttttcatctttcccctgcacttatgttactattct ctgctcccagccttcatccttttctaaaaaggagcaaattctcactctaggctttatc gtgtttactttttcattacacttgacttgattttctagttttctatacaaacaccaat gggttccatctttctgggctcctgattgctcaagcacagtttggcctgatgaagagga tttcaactacacaatactatcattgtcaggactatgacctcaggcactctaaacatat gttttgtttggtcagcacagcgtttcaaaaagtgaagccactttataaatatttggag attttgcaggaaaatctggatccccaggtaaggatagcagatggttttcagttatctc cagtccacgttcacaaaatgtgaaggtgtggagacacttacaaagctgcctcacttct cactgtaaacattagctctttccactgcctacctggaccccagtctaggaattaaatc tgcacctaaccaaggtcccttgtaagaaatgtccattcaagcagtcattctctgggta tataatatgattttgactaccttatctggtgttaagatttgaagttggccttttattg gactaaaggggaactcctttaagggtctcagttagcccaagtttcttttgcttatatg ttaatagttttaccctctgcattggagagaggagtgctttactccaagaagctttcct catggttaccgttctctccatcatgccagccttctcaacctttgcagaaattactaga gaggatttgaatgtgggacacaaaggtcccatttgcagttagaaaatttgtgtccaca aggacaagaacaaagtatgagctttaaaactccataggaaacttgttaatcaacaaag aagtgttaatgctgcaagtaatctcttttttaaaactttttgaagctacttattttca gccaaataggaatattagagagggactggtagtgagaatatcagctctgtttggatgg tggaaggtctcattttattgagatttttaagatacatgcaaaggtttggaaatagaac ctctaggcaccctcctcagtgtgggtgggctgagagttaaagacagtgtggctgcagt agcatagaggcgcctagaaattccacttgcaccgtagggcatgctgataccatcccaa tagctgttgcccattgacctctagtggtgagtttctagaatactggtccattcatgag atattcaagattcaagagtattctcacttctgggttatcagcataaactggaatgtag tgtcagaggatactgtggcttgttttgtttatgtttttttttcttattcaagaaaaaa gaccaaggaataacattctgtagttcctaaaaatactgacttttttcactactataca taaagggaaagttttattcttttatggaacacttcagctgtactcatgtattaaaata ggaatgtgaatgctatatactctttttatatcaaaagtctcaagcacttatttttatt ctatgcattgtttgtcttttacataaataaaatgtttattagattgaataaagcaaaa t act caggt gagcat cctgcctcctgttcccattcctagtagctaaa ggagagggagaaggctctcgggcggagagaggtcctgcccagctgttggcgaggagtt Human TGF- tcctgtttcccccgcagcgctgagttgaagttgagtgagtcactcgcgcgcacggagc beta receptor gacgacacccccgcgcgtgcacccgctcgggacaggagccggactcctgtgcagcttc type-2 (TGFR2) cctcggccgccgggggcctccccgcgcctcgccggcctccaggccccctcctggctgg transcript cgagcgggcgccacatctggcccgcacatctgcgctgccggcccggcgcggggtccgg variant A agagggcgcggcgcggaggcgcagccaggggtccgggaaggcgccgtccgctgcgctg NCBI Reference ggggctcggtctatgacgagcagcggggtctgccatgggtcgggggctgctcaggggc Sequence: NCBI ctgtggccgctgcacatcgtcctgtggacgcgtatcgccagcacgatcccaccgcacg Reference TGGGGAAGGTCCTGTCCTCCTGGTGACAGTAGTTACGGGTGGAGAAGTGAAGAAGCTG AAGAGAC T AAC CTTTCAGTTTGGTGAT GC AAGAAAGGAC AGT T CTCTCCACATCACTG CaGCCCAGCCTGGTGATACAGGCCTCTACCTCTGTGCAGGAGCTCGCAACTTCAACAA ATTTTACTTTGGATCTGGGACCAAACTCAATGTAAAACCAAATATCCAGAATCCGGAC Cccgcggtatatcaactgcgcgactcaaaatcatccgataagagtgtctgtttgttta ctgacttcgacagtcaaactaatgtctctcagagcaaagattccgatgtct acatcac tgacaagtgcgttctggatatgcggagcatggattttaagtccaactccgccgtagcc tggtccaacaagtcagactttgcctgtgcaaatgctttcaacaactcaatt atccctg aggacactttctttccttcaccggagtcctcatgcgatgttaaactggtcgaaaaatc ttttgagacggatacgaacctcaacttccaaaatttgagcgttattggctttaggatt ctgcttctcaaggttgcggggttcaatctcctgatgacgttgcggctttggagcagct aa ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGCTCCGGGCTTGGTGCTGTCGTCTCTC AACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTC CCTGGACTTTCAAGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTC AT GC T GAT GGC AAC T T C C AAT GAGGGC T C C AAGGC C AC AT AC GAGC AAGGC GT C GAGA AGGAC AAGT T T C T C AT C AAC CAT GC AAGC C T GAC CTTGTCCACTCT GAC AG TGAC C AG TGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGATCTTGGCGGGGGGGC C T T GAGC AGT T CTTCGGGC C AGGGAC AC GGCTCACCGTGC T AGAAGAT C T GAAGAAC G TCTTCCCACCTGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACA GAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATCCCGATCACGTGGAACTGTCT TGGTGGGTCAACGGCAAAGAGGTCCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGA AAGAGC AGC C C GC T C T GAAC GAC AGC AGAT AC T GC C T GAGC AGC AGAC T GAGAGT GT C CGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTCTACGGC C T GAGC GAGAAC GAT GAGT GGAC C C AGGAT AGAGC C AAGC C T GT GAC AC AG AT C GT GT C T GC C GAAGC C T GGGGC AGAGC CGATTGTGGCTTTACCAGC GAGAGC T AC C AGC AGGG CGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCC TCR1 Full GTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGAGGCG sequence with GAAGC GGC GC C AC AAAC TTCTCACTGCT GAAAC AGGC T GGC GAC GT GGAGG AGAAT C C human constant TGGCCCAATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGGGTC (nt) optimized AGC AC C C AGC T GC T GGAGC AGAGC CCTCAGTTTC T AAGC AT C C AAGAGGGAGAAAAT C TCACTGTGTACTGCAACTCCTCAAGTGTTTTTTCCTCCTTACAATGGTATCGACAGGA GCCTGGGGAAGGTCCTGTCCTCCTGGTGACAGTAGTTACGGGTGGAGAAGTGAAGAAG C T GAAGAGAC T AAC CTTTCAGTTTGGTGAT GC AAGAAAGGAC AGT T C T C T C C AC AT C A CTGCAGCCCAGCCTGGTGATACAGGCCTCTACCTCTGTGCCGGAGCTCGCAACTTCAA CAAATTTTACTTTGGATCTGGGACCAAACTCAATGTGAAACCAAATATCCAGAATCCG GACCCCGCGGTATATCAACTGCGCGACTCAAAATCATCCGATAAGAGTGTCTGTTTGT T T AC T GAC T T C GAC AGT C AAAC TAATGTCTCT C AGAGC AAAGAT TCCGATGTCTACAT CACTGACAAATGCGTTCTGGATATGCGGAGCATGGATTTTAAGTCCAACTCCGCCGTA GC C T GGT C C AAC AAGT C AGAC T T T GC C T GT GCAAAT GC T T T C AAC AAC T C AAT TAT C C CTGAGGACACTTTCTTTCCTTCACCGGAGTCCTCATGCGATGTTAAACTGGTCGAAAA ATCTTTTGAGACGGATACGAACCTCAACTTCCAAAATTTGAGCGTTATTGGCTTTCGG ATTCTGCTTCTCAAAGTTGCGGGGTTCAATCTCCTGATGACGTTGCGGCTTTGGAGCA GCTAA

ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGCTCCGGGCTTGGTGCTGTCGTCTCT C AACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTC CCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTC AT GC T GAT GGC AAC T T C C AAT GAGGGC T C C AAGGC C AC AT AC GAGC AAGGC GT C GAGA AGGAC AAGT T T C T C AT C AAC CAT GC AAGC C T GAC CTTGTCCACTCT GAC AG TGAC C AG TGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGATCTTGGCGGGGGGGC CTTGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAgaggacctgaataagg tgttcccccctgaggtggccgtgtttgagccaagcgaggccgagatctcccacaccca gaaggccaccctggtgtgcctggcaaccggcttctttcccgatcacgtggagctgtcc tggtgggtgaacggcaaggaggtgcactctggcgtgtgcacagacccacagcccctga aggagcagcctgccctgaatgattcccgctattgtctgtcctctcggctgagagtgtc tgccaccttttggcagaacccacggaatcacttcagatgccaggtgcagttttacggc ctgtctgagaacgacgagtggacccaggatcgggccaagcctgtgacacagatcgtga gcgcggaagcatggggcagagccgactgtggcttcaccagcgtgtcctatcagcaggg TCR1 Native cgtgctgtccgccaccatcctgtacgagatcctgctgggcaaggccacactgtatgcc full sequence gtgctggtgtctgccctggtgctgatggccatggtgaagagaaaagacttcggctccg with human gagcaaccaatttcagcctgctgaagcaggccggcgatgtggaggagaatcctggccc constant (nt) aATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGGGTGAGCACC C AGC T GC T GGAGC AGAGC CCTCAGTTTC T AAGC AT C C AAGAGGGAGAAAAT C T C AC T G TGTACTGCAACTCCTCAAGTGTTTTTTCCAGCTTACAATGGTACAGACAGGAGCCTGG GGAAGGTCCTGTCCTCCTGGTGACAGTAGTTACGGGTGGAGAAGTGAAGAAGCTGAAG AGACTAACCTTTCAGTTTGGTGATGCAAGAAAGGACAGTTCTCTCCACATCACTGCaG CCCAGCCTGGTGATACAGGCCTCTACCTCTGTGCAGGAGCTCGCAACTTCAACAAATT TTACTTTGGATCT GGGAC C AAAC T C AAT GT AAAAC C AAAT AT C C AGAAT C C GGAC C C C gcggtatatcaactgcgcgactcaaaatcatccgataagagtgtctgtttgtttactg acttcgacagtcaaactaatgtctctcagagcaaagattccgatgtctacatcactga caagtgcgttctggatatgcggagcatggattttaagtccaactccgccgt agcctgg tccaacaagtcagactttgcctgtgcaaatgctttcaacaactcaattatccctgagg acactttctttccttcaccggagtcctcatgcgatgttaaactggtcgaaaaatcttt gactaacctttcagtttggtgatgcaagaaaggacagttctctccacatcactgcagc ccagcctggtgatacaggcctctacctctgtgcaggagctcgcaacttcaacaaattt tactttggatctgggaccaaactcaatgtaaaaccaaatatccagaatccggaccccg cggtatatcaactgcgcgactcaaaatcatccgataagagtgtctgtttgtttactga cttcgacagtcaaactaatgtctctcagagcaaagattccgatgtctacatcactgac aagtgcgttctggatatgcggagcatggattttaagtccaactccgccgtagcctggt ccaacaagtcagactttgcctgtgcaaatgctttcaacaactcaattatccctgagga cactttctttccttcaccggagtcctcatgcgatgttaaactggtcgaaaaatctttt gagacggatacgaacctcaacttccaaaatttgagcgttattggctttaggattctgc ttctcaaggttgcggggttcaatctcctgatgacgttgcggctttggagcagctaa atatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaa gtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggat tctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaaga gcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaa caacagcattattccagaagacaccttcttccccagcccaggtaagggcagctttggt gccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctctggt caatgatgtctaaaactcctctgattggtggtctcggccttatccattgccaccaaaa ccctctttttactaagaaacagtgagccttgttctggcagtccagagaatgacacggg aaaaaagcagatgaagagaaggtggcaggagagggcacgtggcccagcctcagtctct ccaactgagttcctgcctgcctgcctttgctcagactgtttgccccttactgctcttc taggcctcattctaagccccttctccaagttgcctctccttatttctccctgtctgcc aaaaaatctttcccagctcactaagtcagtctcacgcagtcactcattaacccaccaa tcactgattgtgccggcacatgaatgcaccaggtgttgaagtggaggaattaaaaagt cagatgaggggtgtgcccagaggaagcaccattctagttgggggagcccatctgtcag ctgggaaaagtccaaataacttcagattggaatgtgttttaactcagggttgagaaaa cagctaccttcaggacaaaagtcagggaagggctctctgaagaaatgctacttgaaga taccagccctaccaagggcagggagaggaccctatagaggcctgggacaggagctcaa tgagaaaggagaagagcagcaggcatgagttgaatgaaggaggcagggccgggtcaca gggccttctaggccatgagagggtagacagtattctaaggacgccagaaagctgttga tcggcttcaagcaggggagggacacctaatttgcttttcttttttttttttttttttt tttttttttttgagatggagttttgctcttgttgcccaggctggagtgcaatggtgca tcttggctcactgcaacctccgcctcccaggttcaagtgattctcctgcctcagcctc ccgagtagctgagattacaggcacccgccaccatgcctggctaattttttgtattttt Human TCR agtagagacagggtttcactatgttggccaggctggtctcgaactcctgacctcaggt alpha constant gatccacccgcttcagcctcccaaagtgctgggattacaggcgtgagccaccacaccc (TRAC) ggcctgcttttcttaaagatcaatctgagtgctgtacggagagtgggttgtaagccaa NCBI Reference gagtagaagcagaaagggagcagttgcagcagagagatgatggaggcctgggcagggt Sequence: ggtggcagggaggtaaccaacaccattcaggtttcaaaggtagaaccatgcagggatg NG_001332.3, agaaagcaaagaggggatcaaggaaggcagctggattttggcctgagcagctgagtca TRAC atgatagtgccgtttactaagaagaaaccaaggaaaaaatttggggtgcagggatcaa aactttttggaacatatgaaagtacgtgtttatactctttatggcccttgtcactatg tatgcctcgctgcctccattggactctagaatgaagccaggcaagagcagggtctatg tgtgatggcacatgtggccagggtcatgcaacatgtactttgtacaaacagtgtatat tgagtaaatagaaatggtgtccaggagccgaggtatcggtcctgccagggccaggggc tctccctagcaggtgctcatatgctgtaagttccctccagatctctccacaaggaggc atggaaaggctgtagttgttcacctgcccaagaactaggaggtctggggtgggagagt cagcctgctctggatgctgaaagaatgtctgtttttccttttagaaagttcctgtgat gtcaagctggtcgagaaaagctttgaaacaggtaagacaggggtctagcctgggtttg cacaggattgcggaagtgatgaacccgcaataaccctgcctggatgagggagtgggaa gaaattagtagatgtgggaatgaatgatgaggaatggaaacagcggttcaagacctgc ccagagctgggtggggtctctcctgaatccctctcaccatctctgactttccattcta agcactttgaggatgagtttctagcttcaatagaccaaggactctctcctaggcctct gtattcctttcaacagctccactgtcaagagagccagagagagcttctgggtggccca gctgtgaaatttctgagtcccttagggatagccctaaacgaaccagatcatcctgagg acagccaagaggttttgccttctttcaagacaagcaacagtactcacataggctgtgg gcaatggtcctgtctctcaagaatcccctgccactcctcacacccaccctgggcccat attcatttccatttgagttgttcttattgagtcatccttcctgtggtagcggaactca ctaaggggcccatctggacccgaggtattgtgatgataaattctgagcacctacccca tccccagaagggctcagaaataaaataagagccaagtctagtcggtgtttcctgtctt gaaacacaatactgttggccctggaagaatgcacagaatctgtttgtaaggggatatg cacagaagctgcaagggacaggaggtgcaggagctgcaggcctcccccacccagcctg