Modified immune cells having enhanced function and methods for screening for same
View Patent ↗The present disclosure provides gene edited modified immune cells or precursors thereof (e.g., gene edited modified T cells) comprising an exogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR) having specificity for a target antigen, and an insertion and/or deletion in one or more endogenous gene loci, wherein the endogenous gene loci encode regulators of T cell function, thereby resulting in immune cells having enhanced function. Compositions and methods of treatment are also provided. The present invention provides methods of screening for TCR- or CAR-T cells with enhanced immune function (e.g., T cell efficacy, T cell memory, and/or T cell persistence).
1. A modified immune cell or precursor cell thereof, comprising:
(A) an insertion and/or deletion in a gene locus encoding for a transcriptional modulator, wherein the insertion and/or deletion downregulates expression of the endogenous transcriptional modulator, wherein the transcriptional modulator is:
(i) a transcription factor, SIX Homeobox 2 (SIX2) or Krüppel-like factor 4 (KLF4), wherein the insertion and/or deletion in the gene locus encoding for SIX2 or KLF4 downregulates gene expression of SIX2 or KLF4, and optionally downregulates gene expression of one or more downstream targets of SIX2 or KLF4 or
(ii) an epigenetic regulator, histone-lysine-N-methyltransferase 2D (KMT2D) or PAXIP1 Associated Glutamate Rich Protein 1 (PAGR1); and
(B) an exogenous T cell receptor (TCR) and/or chimeric antigen receptor (CAR) comprising affinity for an antigen on a target cell.
2. The modified cell of claim 1 , optionally wherein:
the insertion and/or deletion in a gene locus is mediated by a CRISPR-related system; and/or
the insertion and/or deletion in a gene locus is mediated by CRISPR/Cas9.
3. The modified cell of claim 1 , wherein the insertion and/or deletion in the gene locus encoding for PAGR1 downregulates gene expression of one or more downstream targets of the PAGR1-associated histone methyltransferase complex, optionally 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.
4. The modified cell of claim 1 , optionally wherein:
the exogenous TCR is selected from the group consisting of a wild-type TCR, a high affinity TCR, and a chimeric TCR;
the exogenous TCR comprises at least one disulfide bond;
the exogenous TCR comprises a TCR alpha chain and a TCR beta chain; and/or
the exogenous CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, optionally further comprising a hinge domain, optionally 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, optionally 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 intracellular domain comprises at least one co-stimulatory domain selected from 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 comprises an intracellular domain 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, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
5. The modified cell of claim 1 , further comprising an insertion and/or deletion in one or more gene loci encoding for a protein selected from the group consisting of AZI2, C1orf141, CCDC33, CCL7, CEACAM19, MFSD5, and USP27X, wherein the insertion and/or deletion downregulates gene expression of the one or more endogenous genes.
6. The modified cell of claim 1 , optionally wherein:
the antigen on a target cell is a tumor associated antigen (TAA);
the modified cell is an autologous cell;
the modified cell is derived from a human; and/or
the modified cell is a modified T cell.