Engineered Genetic Circuits Activated by Bezafibrate Improve ESC-Based TAA Cancer Vaccine Efficacy and PD-L1 Nanobody Therapy

Abstract

Immunotherapy targeting tumor antigens and immune checkpoint inhibitors has garnered significant attention in cancer treatment. Synthetic gene circuits are developed, encoded in plasmids, which regulate the expression of tumor antigens shared with embryonic stem cells (ESCs) and PD-L1 nanobody (PD-L1 nb) in response to bezafibrate stimulation. This approach significantly minimizes side effects and improved therapeutic efficacy. The transcriptional switches leverage the interaction between the bezafibrate-responsive transcriptional activator PPARγ and RXRα, which are fused with the VPR/VP64/p65 activation domains (AD) and the Gal4 DNA-binding domain (DBD), respectively. These synthetic constructs are validated and their ability to modulate gene expression upon bezafibrate treatment are confirmed. Notably, the gene expression is precise and tunable in response to bezafibrate administration. HEK293T cells or ESCs are employed to deliver this gene circuit, or the plasmids containing the circuit into the tumor are directly injected. Administration of bezafibrate reduces tumor growth, increases specific CD8⁺ T cells, and mitigates CD8⁺ T cell exhaustion, underscoring the feasibility and effectiveness of the approach. ESC-based and intratumoral delivery of the synthetic gene circuits and cargo genes, particularly PD-L1 nb, significantly inhibit tumor growth. PD-L1 nb effectively blocks PD-L1 expression both in vitro and in vivo, as confirmed by using a mutant PD-L1 nb sequence.