Gene-regulating compositions and methods for improved immunotherapy
The present disclosure provides methods and compositions related to the modification of immune effector cells to increase therapeutic efficacy. In some embodiments, immune effector cells modified to reduce expression of one or more endogenous target genes, or to reduce one or more functions of an endogenous protein to enhance effector functions of the immune cells are provided. In some embodiments, immune effector cells further modified by introduction of transgenes conferring antigen specificity, such as exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) are provided. Methods of treating a cell proliferative disorder, such as a cancer, using the modified immune effector cells described herein are also provided.
1. A primary human T cell comprising (a) a modified endogenous ZC3H12A gene and (b) an engineered antigen receptor that specifically binds to a tumor antigen,
wherein ZC3H12A protein expression and/or function is reduced in the primary human T cell, relative to ZC3H12A protein expression and/or function in a primary human T cell expressing the endogenous ZC3H12A gene.
2. The primary human T cell of claim 1 , further comprising a modified endogenous target gene selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, BCOR, BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, MAP4K1 and NR4A3.
3. The primary human T cell of claim 1 , wherein the modified endogenous ZC3H12A gene comprises an insertion, deletion, and/or mutation.
4. The primary human T cell of claim 1 , further comprising a gene-regulating system.
5. The primary human T cell of claim 1 , wherein the primary human T cell is a tumor infiltrating lymphocyte.
6. The primary human T cell of claim 1 , further comprising an exogenous transgene expressing an immune activating molecule selected from the group consisting of a cytokine, a chemokine, a co-stimulatory molecule, an activating peptide, and an antibody or an antigen-binding fragment thereof.
7. A pharmaceutical composition comprising the primary human T cell of claim 1 .
8. The primary human T cell of claim 1 , wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).
9. The primary human T cell of claim 4 , wherein the gene regulating system comprises a Cas endonuclease and a guide RNA (gRNA).
10. The primary human T cell of claim 9 , wherein the Cas endonuclease is:
(a) a wild-type Cas protein comprising two enzymatically active domains;
(b) a Cas nickase mutant comprising one enzymatically active domain;
(c) a deactivated Cas protein (dCas) associated with a heterologous protein; or
(d) a Cas ortholog.
11. The primary human T cell of claim 10 , wherein the Cas endonuclease is a Cas9 protein.
12. The primary human T cell of claim 9 , wherein the gRNA comprises a targeting domain sequence that hybridizes to a target sequence in the endogenous ZC3H12A gene.
13. The primary human T cell of claim 12 , wherein the target sequence is selected from the group consisting of SEQ ID NOs: 1065-1264.
14. The primary human T cell of claim 12 , wherein the gRNA targeting domain sequence is encoded by a sequence selected from the group consisting of SEQ ID NOs: 1065-1264.
15. The primary human T cell of claim 4 , wherein the gene regulating system comprises (a) a zinc finger nuclease that comprises a zinc finger binding domain or (b) a transcription-activator-like effector nuclease (TALEN) that comprises a transcription-activator-like (TAL) effector domain.
16. The primary human T cell of claim 15 , wherein the zinc finger binding domain or TAL effector domain binds to a target sequence in the endogenous ZC3H12A gene.
17. The primary human T cell of claim 16 , wherein the zinc finger binding domain or TAL effector domain binds to a target sequence selected from the group consisting of SEQ ID NOs: 1065-1264.
18. The primary human T cell of claim 4 , wherein the gene regulating system comprises an RNA interference molecule.
19. The primary human T cell of claim 18 , wherein the RNA interference molecule is an siRNA, an shRNA, a microRNA (miR), an antagomiR, or an antisense RNA.
20. The primary human T cell of claim 19 , wherein the RNA interference molecule is an siRNA or shRNA that binds to a sequence encoded by a sequence selected from the group consisting of SEQ ID NOs: 1065-1264.
21. The primary human T cell of claim 1 , wherein the primary human T cell is selected from CD4+ T cells, CD8+ T cells, Th1 cells, Th2 cells, and Th17 cells.