The establishment of polypeptide PSMA‐targeted chimeric antigen receptor‐engineered natural killer cells for castration‐resistant prostate cancer and the induction of ferroptosis‐related cell death

BACKGROUND: The mortality of castration‐resistant prostate cancer (CRPC) is high due to lack of an effective treatment. Chimeric antigen receptor (CAR)‐based therapy is a promising immunotherapeutic strategy. Here, we aimed to design a novel CAR‐natural killer (NK) cells with a clinically significant tumoricidal effect on CRPC. METHODS: We constructed novel CAR‐NK92MI cells with a CD244‐based recombinant lentiviral vector. Different intracellular segments (CD244, NKG2D, or CD3ζ) were screened to identify the best candidate according to cell lysis assay and CD107a expression levels. To enhance the affinity of the CAR to the tumor antigen, we compared an antibody specific for prostate‐specific membrane antigen (anti‐PSMA) with PSMA‐targeted polypeptide (p‐PSMA), which was screened by phage display combinatorial library. Then, CAR‐NK92MI cells with both a high affinity for PSMA and a strong tumoricidal capacity were generated. In addition, we verified their tumor‐killing effect in vitro and in vivo. The release of cytokine by NK92MI cells was compared with that by CAR‐NK92MI cells through flow cytometry and enzyme‐linked immunosorbent assay. Moreover, ferroptosis‐related cell death was explored as a possible underlying mechanism. RESULTS: Three different CAR intracellular regions CAR1 (CD244), CAR2 (CD244, NKG2D) and CAR3 (CD244, NKG2D, and CD3ζ) were constructed. CAR2 was chosen to confer a stronger tumoricidal ability on CAR‐NK92MI cells. Compared with anti‐PSMA, p‐PSMA exhibited enhanced affinity for the tumor antigen. Thus, p‐PSMA‐CAR‐NK92MI cells, which expressed CAR with a polypeptide‐based antigen‐binding region, an intracellular CD244 and a NKG2D costimulatory domain, were generated. They could selectively and successfully kill PSMA(+) target cells and exhibited specific lysis rate of 73.19% for PSMA‐positive C4‐2 cells and 33.04% for PSMA‐negative PC3 cells. Additionally, p‐PSMA‐CAR‐NK92MI cells had significantly higher concentrations of IFN‐γ, TNF‐α and granzyme B than NK92MI cells. In a CRPC cancer xenograft model, p‐PSMA‐CAR‐NK92MI cells significantly inhibited tumor growth and exerted a more consistent killing effect than NK92MI cells. Moreover, ferroptosis is a potential mechanism through which CAR‐NK92MI cells attack cancer cells, and is triggered by IFN‐γ. CONCLUSIONS: p‐PSMA‐CAR‐NK92MI cells can effectively kill CRPC(PSMA+) cells in vitro and in vivo. This strategy may provide additional treatment options for patients with CRPC.