NK cells coexpressing KIR and NKG2C as a basis for HER2-targeted CAR-NK effectors

Background. For the development of HER2-targeted CAR NK cell effectors, it is important to select an NK cells subset with high cytotoxic potential and sensitivity to genetic modification. In the context of solid tumor therapy, licensed KIR⁺ NK cells expressing the activating receptor NKG2C are of particular interest.

Aim. To obtain HER2-CAR-NK cells based on NK cell subsets with different expression of KIR and NKG2C receptors for the elimination of HER2⁺ solid tumors.

Materials and methods. NK cells from an HCMV-seropositive donor were isolated by negative magnetic separation from peripheral blood mononuclear cells isolated by density gradient centrifugation. NK cell subsets with different expression of KIR and NKG2C were obtained by cell sorting and cultured with stimulation by IL-2 and feeder cells. NK cells were modified with a gene carrying the HER2-CAR construct via retroviral transduction. The cytotoxic activity of the cultures against SKOV-3 spheroids was detected by the level of degranulating CD107a⁺ NK cells and the proportion of nonviable SytoxBlue⁺ tumor cells in the spheroid.

Results. HER2-CAR-NK effectors were obtained based on subsets with different KIR and NKG2C expression. It was shown that NKG2C-expressing NK cells, regardless of KIR expression, have greater sensitivity to retroviral transduction. Upon co-incubation of the obtained cultures of NK cell subsets with HER2⁺ SKOV-3 tumor cell spheroids, in the fraction of transduced cells bearing the HER2-CAR receptor, the proportion of degranulated CD107a⁺ cells was more than 4-fold higher than the level of degranulation in the fraction of unmodified NK cells. Also, in SKOV-3 spheroids after co-incubation with KIR⁺ NKG2C⁺ transduced NK effectors, a lower proportion of viable cells was observed compared to spheroids that were incubated with KIR⁺ NKG2C^– cultures.

Conclusion. NKG2C+ NK cells exhibited greater sensitivity to transduction. Cultures of KIR⁺NKG2C⁺ HER2-CAR-NK cells eliminated HER2⁺ target cells more efficiently in a 3D model. These properties make NK cells coexpressing KIR and NKG2C a promising basis for the CAR-NK effectors development.

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