Abstract: Objective To obtain novel dual-effect cord blood natural killer cells (CBNKCs) expressing high-affinity PD-1 (HAPD1) and chimeric antigen CD19 receptor (CAR) to improve the effect of CAR-based immunotherapy. Methods A dual-effect lentiviral vector expressing both HAPD1 and CAR targeting CD19 was constructed. CBNKCs were infected withthe vector to obtain HAPD1 CAR19 CBNKCs. The surface markers of the cells including CD3-/CD16+CD56+, CD3+/CD16+CD56+,CD3+/CD4+, and CD3+/CD8+ were tested during cell proliferation. The cytotoxicity of CBNKCs, CAR19 CBNKCs and HAPD1 CAR19 CBNKCs incubated with CD19-positive target cells at the effector-target ratios of 5∶1, 10∶1 and 20∶1 was tested on days 7, 9, 12, and 15 of cell culture. The cytotoxicity of the cells against the target cells was also tested in NPG mice. Results CBNKCs were successfully transduced with T-cell designed CAR19 and HAPD1 CAR19 with an efficiency of (18.63±1.88)%. Infection with the lentiviral vector significantly reduced the cell expansion efficiency of the CBNKCs (10.97±2.77 vs 24.84±3.17, P<0.05) but did not significantly affect the expressions of the surface markers (P>0.05). HAPD1 CAR CBNKCs showed stronger anti-tumor effect than CAR19 CBNKCs [(68.38±8.08)% vs (49.65±13.60)% at the effector-target ratios of 5∶1 and (79.11±7.42)% vs (59.78 ± 9.32)% at 10∶1; P<0.05]. The infected CBNKCs showed the strongest cytotoxicity at 9 and 12 days after lentivirus infection. In the mouse models, transplantation of the dual-effect cells resulted in a significantly lower percentage of tumor cells in white blood cells than transplantation CAR-CBNKCs [(19.21 ± 3.07% ) vs (29.08 ± 3.15)% , P<0.05]. Conclusion We obtained a novel dual-effect CBNKC co-expressing HAPD1 and CAR. The cells show strong cytotoxicity against the target tumor cells both in vitro and in vivo, which sheds light on a new strategy of immunotherapy against tumor cells.

Key words: chimeric antigen receptor; cord blood NK cells; high-affinity PD-1; immunophenotypes; antitumor efficacy