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 SEQ ID NO:63, including 100% identity in the binding domain comprised therein, wherein the binding domain comprises SEQ ID NO: 81.
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.