NR2F6 INHIBITED CHIMERIC ANTIGEN RECEPTOR CELLS
Disclosed are compositions of matter, cells, and methodologies for generation of chimeric antigen receptor (CAR) cells with inhibited or absent NR2F6 activity. In one embodiment, a CAR possessing affinity to a tumor antigen is transfected onto T cells that possess reduced or absent NR2F6 activity, said reduction or absence of NR2F6 activity leading to increased production of cytokines associated with inhibition of tumor growth, metastasis or angiogenesis, and/or augmentation of tumor cytotoxicity. Inhibition of NR2F6 activity may be performed ex vivo on said T cells or in vivo by administration of small molecule inhibitors, siRNA, shRNA or gene editing. In some embodiments other immune cells are substituted for CAR-T cells.
1 . A method of treating cancer comprising the steps of: a) obtaining a cell population from peripheral blood; b) transfecting said population with a chimeric antigen receptor (CAR); c) introducing said transfected cell population into said patient; and d) inhibiting NR2F6 activity to an extent sufficient to enhance T cell activation.
2 . The method of claim 1 , wherein said blood cell population is selected from a group comprising of: a) peripheral blood mononuclear cells; b) CD4 T cells; c) CD8 T cells; d) NK cells; e) NKT cells; and f) gamma delta T cells.
3 . The method of claim 2 , wherein said CD4 T cells are isolated by means of magnetic separation prior to transfection with CAR.
4 . The method of claim 2 , wherein said CD8 T cells are isolated by means of magnetic separation prior to transfection with CAR.
5 . The method of claim 1 , wherein said CAR is comprised of: a) an antigen binding domain; b) a transmembrane domain; c) a costimulatory signaling region; d) a CD3 zeta signaling domain.
6 . The method of claim 5 , wherein said CD3 zeta chain is resistant to cleavage by caspase 3 by means of amino acid substitution.
7 . The method of claim 5 , wherein the antigen binding domain is an antibody or an antigen-binding fragment thereof.
8 . The method of claim 7 , wherein the antigen-binding fragment is a Fab or a scFv.
9 . The method of claim 5 , wherein the antigen binding domain binds to a tumor specific and/or tumor associated antigen.
10 . The method of claim 9 , wherein said tumor specific and/or tumor associated antigen is selected from a group of antigens comprising of: a) HER2; b) CD19; c) EGFR; d) CD20; e) MUC1; and f) CD105.
11 . The method of claim 5 , wherein said costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group comprising of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
12 . The method of claim 1 , wherein said transfected cell population is allogeneic to the cancer patient in need of treatment.
13 . The method of claim 1 , wherein said transfected cell population is autologous to the cancer patient in need of treatment.
14 . The method of claim 1 , wherein an inhibitor of a CD3 inhibitory molecule is co-administered together with the CAR.
15 . The method of claim 14 , wherein said inhibitor of CD3 inhibitory molecule is a dominant negative CTLA-4.
16 . The method of claim 14 , wherein said inhibitor of CD3 inhibitory molecule is a dominant negative IL-10 receptor.
17 . The method of claim 14 , wherein said inhibitor of CD3 inhibitory molecule is a dominant negative TGF-beta receptor.
18 . The method of claim 1 , wherein said CAR transfected cells are cotransfected with an a molecule capable of inducing RNA interference.
19 . The method of claim 18 , wherein said molecule capable of inducing RNA interference are selected from a group comprising of: a) siRNA or b) shRNA.
20 . The method of claim 19 , wherein silencing of molecules that inhibit CD3 zeta signaling are silenced.
21 . The method of claim 20 , wherein silencing of molecules is achieved, said molecules selected from a group comprising of: a) OX2; b) TGF-beta receptor; c) SMAD4; d) IL-10 receptor; e) PD-1; and f) CTLA-4.
22 . The method of claim 18 , wherein silencing of NR2F6 is achieved through introduction of introducing a short hairpin loop RNA (shRNA) comprising a sense sequence of 5′-GAT CCG CAT TAC GGT GTC TTC ACC TTC AAG AGA GGT GAA GAC ACC GTA ATG CTT TTT TCT AGA G-3′ or a sense sequence of 5′-GAT CCG CCT CTG GAC ACG TAA CCT ATT CAA GAG ATA GGT TAC GTG TCC AGA GGT TTT TTC TAG AG-3′