TY - JOUR
T1 - Homology-independent targeted insertion-mediated derivation of M1-biased macrophages harbouring Megf10 and CD3ζ from human pluripotent stem cells
AU - Zhen, Xing
AU - Kim, Jieun
AU - Kang, Jong Soon
AU - Choi, Byeong Jo
AU - Park, Ki Hwan
AU - Lee, Dong Seok
AU - Hong, Seok Ho
AU - Lee, Jong Hee
N1 - Publisher Copyright:
© 2024 The Author(s)
PY - 2024/11
Y1 - 2024/11
N2 - Background: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified 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 inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), 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 signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids. Interpretation: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours. Funding: This work was supported byKRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and aKorean Fund for Regenerative Medicine (KFRM) grant funded by theKorean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).
AB - Background: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified 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 inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), 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 signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids. Interpretation: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours. Funding: This work was supported byKRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and aKorean Fund for Regenerative Medicine (KFRM) grant funded by theKorean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).
KW - CD3ζ
KW - Chimeric antigen receptor (CAR)-Modified macrophage
KW - Homology-independent targeted insertion (HITI)
KW - Human pluripotent stem cell (hPSC)
KW - Immunotherapy
KW - Megf10
UR - http://www.scopus.com/inward/record.url?scp=85205690667&partnerID=8YFLogxK
U2 - 10.1016/j.ebiom.2024.105390
DO - 10.1016/j.ebiom.2024.105390
M3 - Article
C2 - 39383607
AN - SCOPUS:85205690667
SN - 2352-3964
VL - 109
JO - eBioMedicine
JF - eBioMedicine
M1 - 105390
ER -