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 human tumor infiltrating lymphocyte (TIL) comprising (a) a modified endogenous ZC3H12A gene and (b) a Cas endonuclease and/or a guide RNA (gRNA), wherein
ZC3H12A protein expression and/or function is reduced in the human TIL, relative to ZC3H12A protein expression and/or function in an unmodified human TIL expressing an endogenous ZC3H12A gene, and
anti-tumor efficacy of the human TIL is increased, relative to anti-tumor efficacy of an unmodified human TIL expressing an endogenous ZC3H12A gene.
2. The human TIL of claim 1 , wherein the modified endogenous ZC3H12A gene comprises an insertion, deletion, and/or mutation.
3. The human TIL of claim 1 , 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.
4. The human TIL of claim 3 , wherein the Cas endonuclease is a wild-type Cas9 protein comprising two enzymatically active domains.
5. The human TIL of claim 1 , wherein the gRNA comprises a targeting domain sequence that hybridizes to a target sequence in the endogenous ZC3H12A gene.
6. The human TIL of claim 5 , wherein the target sequence is selected from the group consisting of SEQ ID NOs: 1065-1264.
7. The human TIL of claim 5 , wherein the gRNA targeting domain sequence is encoded by a sequence selected from the group consisting of SEQ ID NOs: 1065-1264.
8. The human TIL 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.
9. The human TIL 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.
10. The human TIL of claim 9 , further comprising a modified endogenous TGFBR2 gene.
11. The human TIL of claim 1 , comprising the Cas endonuclease and the gRNA.
12. The human TIL of claim 11 , wherein the Cas endonuclease is a wild-type Cas9 protein comprising two enzymatically active domains.
13. The human TIL of claim 12 , wherein the gRNA comprises a targeting domain sequence that hybridizes to a target sequence in the endogenous ZC3H12A gene.
14. The human TIL of claim 13 , wherein the target sequence is selected from the group consisting of SEQ ID NOs: 1065-1264.
15. The human TIL of claim 13 , wherein the gRNA targeting domain sequence is encoded by a sequence selected from the group consisting of SEQ ID NOs: 1065-1264.
16. A pharmaceutical composition comprising the human TIL of claim 1 .