Homology-independent targeted insertion-mediated derivation of M1-biased macrophages harbouring Megf10 and CD31; 1; from human pluripotent stem cells

초록

Background Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate fi ltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified fi ed macrophages (CAR-Ms) are challenging. Methods Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity. Findings These engineered cells phagocytosed cancer cells, leading to significant fi cant inhibition of cancer-cell proliferation in vitro and in vivo. . Furthermore, the engineered CARs, which incorporated a combination of CD31; 1; and Megf10 (referred to as FRP5M1;), M 1; ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5M1; M 1; promoted M1 polarisation via nuclear factor kappa B (NF-KB), K B), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory fl ammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration fi ltration of T cells in cancer spheroids. Interpretation Our fi ndings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD31; 1; and Megf10 domains is an effective strategy for the immunotherapy of solid tumours.

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