Modified immune cells having enhanced function and methods for screening for same

1. A method for generating a modified immune cell or precursor cell thereof, comprising:

a) introducing into the immune cell a first nucleic acid comprising a nucleic acid sequence encoding an exogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell; and

b) introducing into the immune cell one or more polypeptides and/or nucleic acids that downregulate a gene expression of an endogenous transcriptional modulator;

wherein the endogenous transcriptional modulator is downregulated with a CRISPR system-mediated insertion and/or deletion in a gene locus encoding for the endogenous transcriptional modulator; and

wherein the endogenous transcriptional modulator is selected from the group consisting of AZI2, Clorf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X.

2. A method for generating a modified immune cell or precursor cell thereof, comprising:

a) introducing into the immune cell a first nucleic acid comprising a nucleic acid sequence encoding an exogenous T cell receptor (TCR) and/or an exogenous chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell; and

b) introducing into the immune cell one or more polypeptides and/or nucleic acids that downregulate a gene expression of an endogenous transcriptional modulator; wherein the endogenous transcriptional modulator is downregulated with an insertion and/or deletion in a gene locus encoding for the endogenous transcriptional modulator; and wherein the endogenous transcriptional modulator is a transcription factor or an epigenetic regulator, wherein the transcription factor is SIX Homeobox 2 (SIX2) or Kruppel-like factor 4 (KLF4), and wherein the epigenetic regulator is a histone-lysine-N-methyltransferase 2D (KMT2D), or PAXIPI Associated Glutamate Rich Protein 1 (PAGR1).

3. The method of claim 2 , wherein the insertion and/or deletion in a gene locus encoding for PAGR1 or KMT2D downregulates the gene expression of (a) PAGR1 or KMT2D, and/or (b) one or more downstream targets of the KMT2D- or PAGR1-associated histone methyltransferase complex.

4. The method of claim 3 , wherein the one or more downstream targets of the PAGR1-associated histone methyltransferase complex is selected from the group consisting of ARID1A, ARID3B, ASXL1, DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, and TNFAIP3.

5. The method of claim 2 , wherein the insertion and/or deletion in the gene locus encoding for the endogenous transcriptional modulator is mediated by:

(a) a CRISPR-related system;

(b) a CRISPR nuclease and a guide RNA; and/or

(c) a CRISPR/Cas9.

6. The method of claim 5 , wherein the guide RNA comprises a guide sequence that is complementary to a target sequence of the endogenous transcriptional modulator.

7. The method of claim 2 , wherein the insertion and/or deletion is in one or more gene loci encoding for the endogenous transcriptional modulator further comprises AZI2, Clorfl41, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2 or USP27X.

8. The method of claim 7 , wherein the endogenous transcriptional modulator is:

(a) the PAGR1 gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 31-36;

(b) the SIX2 gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:37-42;

(c) the Klf4 gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:43-48;

(d) the USP27X gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:49-54;

(e) the CEACAM19 gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:55-60; or

(f) the Clorfl4l gene and the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:61-66.

9. The method of claim 6 , wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 31-66.

10. The method of claim 2 , wherein each of the one or more polypeptides and/or nucleic acids that downregulates gene expression is introduced by electroporation.

11. The method of claim 2 , wherein the first nucleic acid is introduced by viral transduction, and wherein the viral transduction comprises contacting the immune cell or precursor cell thereof with a viral vector.

12. The method of claim 11 , wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

13. The method of claim 12 , wherein the antigen on a target cell is a tumor associated antigen (TAA).

14. The method of claim 12 , wherein the modified cell is:

(a) an autologous cell;

(b) derived from a human; or

(c) a modified T cell.

15. The method of claim 12 , wherein the exogenous TCR

is selected from the group consisting of a wild-type TCR, a high affinity TCR, and a chimeric TCR;

comprises at least one disulfide bond; and/or

comprises a TCR alpha chain and a TCR beta chain.

16. The method of claim 2 , wherein the exogenous CAR comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular domain, wherein:

the antigen-binding domain is selected from the group consisting of an antibody, an scFv, and a Fab;

the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154;

the co-stimulatory domain is selected from at least one of the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3; and/or

the intracellular domain is selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

17. The method of claim 16 , wherein the exogenous CAR further comprises a hinge domain, and wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.

18. A method for generating a modified immune cell or precursor cell thereof, comprising:

a) introducing into the immune cell a first nucleic acid comprising a nucleic acid sequence encoding an exogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell; and

b) introducing into the immune cell one or more polypeptides and/or nucleic acids that downregulate a gene expression of an endogenous transcriptional modulator; wherein the endogenous transcriptional modulator is downregulated with a CRISPR system-mediated insertion and/or deletion in a gene locus encoding for the endogenous transcriptional modulator; and wherein the endogenous transcriptional modulator is:

(i) a transcription factor selected from the group consisting of SIX Homeobox 2 (SIX2) and Krüppel-like factor 4 (KLF4); or

(ii) an epigenetic regulator selected from the group consisting of histone-lysine-N-methyltransferase 2D (KMT2D) or PAXIP1 Associated Glutamate Rich Protein 1 (PAGR1).

19. The method of claim 18 , wherein the insertion and/or deletion in the gene locus encoding for SIX2 or KLF4 downregulates gene expression of SIX2 or KLF4, or one or more downstream targets of SIX2 or KLF4.