Rapid screening of cancer-targeting receptors in natural killer (NK) cells

Science

Autologous chimeric antigen receptor (CAR)-T cell therapy involves the derivation of T cells from a patient, their genetic modification outside of the body with CAR specifically targeting cancer cells and subsequent reinfusion of CAR-T cells into the patient. Despite its remarkable success in treating liquid cancers, CAR-T therapy can result in adverse effects to the patient. Alternative immune cell types such as natural killer (NK) cells can be derived from healthy human donors, engineered to express CAR and administered to many patients in much safer manner that produces little to no side effects. However, modifying NK cells to express CAR is challenging due to the long process time required for and poor efficiency of virus-based CAR delivery. In this study, we developed an optimized non-viral protocol to deliver CAR mRNA payload into NK cells for rapid screening to identify CAR candidates that improve cancer killing by NK cells.

Societal Impact

When implemented, our optimized screening process will reduce the time required for identification of CARs that enhance cancer targeting of NK cells from 2 weeks to 4 days. This will accelerate the development of CAR-NK cell pipelines to target multiple cancer types and progress their timely translation to evaluation in clinical trials.

Technical Summary

We first expanded NK cells from adult peripheral blood using a feeder-based approach. We then tested various conditions that promote non-viral nucleofection- or electroporation-based delivery of CAR mRNA into expanded NK cells. Our empirical iterations eventually shortlisted an optimal nucleofection program which achieved consistently high expression of CAR genes of interest in while preserving viability of NK cells. Using this program, we were able to validate CAR designs expressed in NK cells to target different cancer antigens in a short turnaround period of 4 days, thus corroborating this strategy for efficient screening of CAR candidates.

References

Wang CQ, Leong L, Yang L, Xu S, Lam KP, Choo ABH, and Tan AHM. Nucleofection-based screening of chimeric antigen receptor candidates in human natural killer cells. Front. Immunol. 16, 1557766 (2025).