FIGURE 2.
Screening of CAR constructs that enhanced NK92MI cell activity. (A) Schematic diagram of the structure of three different intracellular segments for screening CAR‐NK92MI cells with the highest antitumor activity. (B) A cell lysis assay showed that NK92MI cells expressing CAR2 or CAR3 exhibited the greatest increases in antitumor activity after coculture with mesohigh SKOV3 target cells at the indicated effector‐to‐target ratios for 4 hours. (C) The CD107a expression assay demonstrated that CAR2‐NK92MI and CAR3‐NK92MI cells showed stronger degranulation than CAR1‐NK92MI. (D) The peptides KHL, WTN and GTI exhibited stronger affinity for PSMA antigen, as demonstrated by immunofluorescence assays. The GTI, KHL, and WTN peptides showed higher affinities for their respective PSMA antigens compared with the HFK, YVN, and WQP peptides. The peptides were edited for green fluorescence. Prostate cancer cells were edited for blue fluorescence. Scale bar: 20 μm. (E) p‐PSMA‐CAR‐NK92MI cells exerted more potent antitumor effects than anti‐PSMA‐CAR‐NK92MI cells according to the cell lysis assay. (F) The CD107a expression assay indicated that p‐PSMA‐CAR‐NK92MI cells exhibited more degranulation after activation by PSMA antigen than anti‐PSMA‐CARNK92MI cells. (G) Flow cytometric results showed that the binding affinity for PSMA antigens of p‐PSMA‐CAR was superior to that of anti‐PSMA scFv. The data are presented as the means ± SEMs from three independent experiments. *, P <0.05; **, P <0.01; ns, not significant; ns, not significant. Abbreviations: CAR, chimeric antigen receptor; KHL, KHLHYHSSVRYG; WTN, WTNHHQHSKVRE; GTI, GTIQPYPFSWGY; PSMA, prostate‐specific membrane antigen; scFv, single‐chain fragment variable; Meso, mesothelin; SEM, standard error of the mean