Chimeric antigen receptor (CAR) and T cell receptor (TCR) modified T cells

1. A zinc finger nuclease (ZFN) comprising:

a first zinc finger protein (ZFP) that binds a first target site in a T-cell receptor alpha constant (TRAC) gene, the first ZFP comprising three or more zinc finger domains;

a second ZFP that binds a second target site in the TRAC gene, the second ZFP comprising three or more zinc finger domains; and

a cleavage domain;

wherein:

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5-7, 2, and 19, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 1-3 and 22, from N-terminus to C-terminus of the second ZFP;

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOS.: 2, 5-7, 2, and 19, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 1-3, and 22, from N-terminus to C-terminus of the second ZFP;

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5, 11, 7, 2, and 9, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 1-3, and 22, from N-terminus to C-terminus of the second ZFP;

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5, 11, 7, 2, and 9, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 1-3, and 22, from N-terminus to C-terminus of the second ZFP;

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5, 15, 7, 2, and 9, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 1-3 and 22, from N-terminus to C-terminus of the second ZFP;

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5, 15, 7, 2, and 9, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 1-3, 22, from N-terminus to C-terminus of the second ZFP; or

the first ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 2, 5, 23, and 7, 2, and 9, from N-terminus to C-terminus of the first ZFP, and the second ZFP comprising amino acid sequences in the following order: SEQ ID NOs: 1-3, 22, from N-terminus to C-terminus of the second ZFP.

2. The ZFN of claim 1 , wherein the TRAC gene is a human TRAC gene.

3. The ZFN of claim 1 , wherein the cleavage domain comprises a wild-type or engineered Fokl cleavage domain.

4. A polynucleotide encoding the ZFN of claim 1 .

5. A vector comprising the polynucleotide of claim 4 .

6. The vector of claim 5 , wherein the vector is an adenoviral or lentiviral vector.

7. A cell or a cell line comprising the ZFN of claim 1 .

8. The cell or the cell line of claim 7 , wherein the cell is a stem cell, a T cell or a Natural Killer (NK) cell.

9. The cell or the cell line of claim 8 , wherein the cell is a T cell derived from a primary human T cell from a human donor.

10. The cell or the cell line of claim 9 , wherein the cell has a reduced expression of endogenous TRAC gene.

11. A cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein a TRAC gene is inactivated using the ZFN of claim 1 .

12. The cell of claim 11 , wherein the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain.

13. The cell of claim 11 , wherein the cell has a reduced graft-versus-host disease (GVHD) response in a bioincompatible human recipient as compared to the GVHD response of the primary human T cell in response to allogenic CAR T treatment.

14. The cell of claim 11 , wherein an antigen binding domain of the CAR binds FZD10, TSHR, PRLR, Muc17, GUCY2C, CD207, CD19, or CD20.

15. The cell of claim 11 , wherein a costimulatory signaling region of the CAR comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

16. The cell of claim 11 , wherein the cell is a T cell comprising a nucleic acid encoding hTERT or a nucleic acid encoding SV40LT, or a combination thereof.

17. A method of treating cancer in a subject, the method comprising

administering the cell of claim 11 to the subject, wherein the cancer is selected from the group consisting of a lung carcinoma, pancreatic cancer, liver cancer, bone cancer, breast cancer, colorectal cancer, leukemia, ovarian cancer, lymphoma, and brain cancer.