Activity and toxicity of human mesenchymal stem cells derived from anti-PD1-integrated iPSCs.

Abstract

e14520 Background: Despite the effectiveness of PD-1 blockade in various cancers, limitations such as poor tumor penetration of aPD-1 antibodies and inability to fully reverse the immunosuppressive tumor microenvironment remain. Systemic aPD-1 antibody administration also risks immune system imbalance and severe toxicities. Here, we propose a strategy for aPD-1 antibody delivery using mesenchymal stem cells (MSCs). Human iPSC-derived MSCs (aPD1-iMSCs) were engineered to stably secrete aPD1-scFv and their anti-tumor efficacy were evaluated in preclinical animal models. Methods: First, we integrated the aPD1-scFv into the safe harbor locus ( B2M ) of human iPSCs and directed their differentiation to obtain aPD1-iMSCs. We then evaluated the anti-tumor efficacy and biodistribution of aPD1-iMSCs in a moderately PD1 responsive mouse model (CT26 colorectal cancer). Multicolor flow cytometry and single-cell RNA sequencing were used to analyze the impact of aPD1-iMSCs on the immune landscape in the spleen and tumor. Results: In vitro, aPD1-iMSCs secreted aPD1-scFv (1.6 µg/10 6 cells/24 h) and effectively blocked the binding of hPD-1 to hPD-L1. When co-cultured with T cells, aPD1-iMSCs demonstrated enhanced survival compared to wild-type iMSCs, indicating lower immunogenicity. In vivo, aPD1-iMSCs localized to the tumor and spleen, exhibiting tumor-suppressive effects comparable to Pembrolizumab (5mg/kg). Furthermore, aPD1-iMSCs increased infiltration of T cells and NK cells into tumor tissue and decreased tumor-associated macrophage infiltration. Notably, aPD1-iMSCs did not induce abnormal peripheral immune cell activation or expansion, unlike Pembrolizumab, suggesting a lower risk of systemic side effects. Conclusions: aPD1-iMSCs effectively home to tumor tissues, modulate the tumor microenvironment, and exhibit significant anti-tumor activity. These findings support the further development of aPD1-iMSCs as a promising, safer approach for immune checkpoint blockade cell therapy.