CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19⁺HLA-C1^- Malignant B Cells While Sparing CD19⁺HLA-C1⁺ Healthy B Cells

B cell aplasia caused by "on-target off-tumor" toxicity is one of the clinical side effects during CD19-targeted chimeric antigen receptor (CAR) T (CD19-CAR-T) cells treatment for B cell malignancies. Persistent B cell aplasia was observed in all patients with sustained remission, which increased the patients' risk of infection. Some patients even died due to infection. To overcome this challenge, the concept of incorporating an inhibitory CAR (iCAR) into CAR-T cells was introduced to constrain the T cells response once an "on-target off-tumor" event occurred. In this study, we engineered a novel KIR/PD-1-based inhibitory CAR (iKP CAR) by fusing the extracellular domain of killer cell immunoglobulin-like receptors (KIR) 2DL2 (KIR2DL2) and the intracellular domain of PD-1. We also confirmed that iKP CAR could inhibit the CD19 CAR activation signal via the PD-1 domain and CD19-CAR-T cells bearing an iKP CAR (iKP-19-CAR-T) exerted robust cytotoxicity in vitro and antitumor activity in the xenograft model of CD19⁺HLA-C1^- Burkitt's lymphoma parallel to CD19-CAR-T cells, whilst sparing CD19⁺HLA-C1⁺ healthy human B cells both in vitro and in the xenograft model. Meanwhile, iKP-19-CAR-T cells exhibited more naïve, less exhausted phenotypes and preserved a higher proportion of central memory T cells (T_CM). Our data demonstrates that the KIR/PD-1-based inhibitory CAR can be a promising strategy for preventing B cell aplasia induced by CD19-CAR-T cell therapy.