FC-EPSILON CAR
Recombinant NK cells, and especially recombinant NK-92 cells express a chimeric antigen receptor (CAR) having an intracellular domain of FcεRIγ. Notably, CAR constructs with an intracellular domain of FcεRIγ had a substantially prolonged duration of expression and significantly extended cytotoxicity over time. The CAR may be expressed from RNA and DNA, preferably as a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK/NK-92 cells.
1 . A genetically modified NK-92 cell, comprising:
a recombinantly expressed cytokine;
a recombinantly expressed CD16;
a membrane bound chimeric antigen receptor (CAR) that comprises a FcεRIγ signaling domain,
wherein the CAR has at least 90% sequence identity to a polypeptide encoded by a nucleic acid having sequence SEQ ID NO:41, including 100% identity in the binding domain comprised therein, wherein the binding domain comprises a scFv portion that binds to CD123.
2 . The genetically modified NK-92 cell of claim 1 , wherein the recombinantly expressed cytokine is IL-2, optionally comprising an endoplasmic retention sequence.
3 . The genetically modified NK-92 cell of claim 1 , wherein the recombinantly expressed cytokine is IL-15, optionally comprising an endoplasmic retention sequence.
4 . The genetically modified NK-92 cell of claim 1 , wherein the recombinantly expressed CD16 has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 35.
5 . The genetically modified NK-92 cell of claim 1 , wherein the FcεRIγ signaling domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1.
6 . The genetically modified NK-92 cell of claim 1 , wherein the FcεRIγ signaling domain has an amino acid sequence of SEQ ID NO:1.
7 . The genetically modified NK-92 cell of claim 1 , wherein the genetically modified NK cell comprises a tricistronic nucleic acid sequence comprising a sequence encoding the recombinantly expressed cytokine, a sequence encoding the recombinantly expressed CD16, and a sequence encoding the recombinantly expressed CAR.
8 . The genetically modified NK-92 cell of claim 7 , wherein the tricistronic nucleic acid sequence is integrated into the genome of the NK-92 cell.
9 . A recombinant nucleic acid encoding a CAR that has at least 90% sequence identity to a polypeptide encoded by a nucleic acid having sequence SEQ ID NO:41, including 100% identity in a binding domain comprised therein, wherein the binding domain comprises a scFv portion that binds to CD123.
10 . The recombinant nucleic acid of claim 9 , wherein the recombinant nucleic acid encodes a CAR that has at least 95% sequence identity to the polypeptide encoded by a nucleic acid having sequence SEQ ID NO:41.
11 . The recombinant nucleic acid of claim 9 , wherein the recombinant nucleic acid has a nucleic acid having sequence SEQ ID NO:41.
12 . A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the genetically modified NK cells of claim 1 , thereby treating the cancer.
13 . The method of claim 12 further comprising a step of administering at least one additional therapeutic entity selected from the group consisting of a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and a tumor targeted cytokine.
14 . The method of claim 12 , wherein the cancer is selected from leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic leukemias, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.