Transient intracellular expression of PD-L1 and VEGFR2 bispecific nanobody in cancer cells inspires long-term T cell activation and infiltration to combat tumor and inhibit cancer metastasis

Abstract

Background: PD-L1, an immune checkpoint inhibitor, and VEGFR2, essential for cancer metastasis, play pivotal roles in tumorigenesis. However, their miniature bispecific intracellular nanobodies for combining check-point blockade and anti-metastasis anticancer therapy remain underexplored. Methods: The intrabodies were developed using gene cloning technology. Specificity of the intrabodies was testified using Western blot, co-immunoprecipitation (co-IP) analysis, antibody competitive binding assay, flow cytometry analysis, etc. Checkpoint blockade was demonstrated using antibody-antigen competitive binding assay. Cancer cell migration was determined using scratch assay. Combined anti-cancer therapeutic efficacy of FAP1V2 was determined in vivo of mice models. The PD-1hi immune cells, TCR βhi and CD25hi T-cells were analyzed by flow cytometry, and cancer cell metastasis was performed using immune-fluorescence analysis on lung and liver tissues. Transcriptome analysis was performed to explore signaling pathways associated with the enhanced anticancer efficiency. Results: Bispecific intrabody FAP1V2 fused with antibody VH regions, was successfully developed and verified with its ability to target and block human and mouse PD-L1 and VEGFR2, inhibiting cancer cell binding to PD-1 and reducing their migratory capacity. Compared to the other treatment, two-rounds of transient FAP1V2 expression in LLC cells in experimental mice models achieved remarkable tumor inhibition, which brought about complete immune inhibition on growth of secondary-round of LLC tumor in 1/6 of the tested mice, inspired long-term activation of TCR βhi T cells and increased their infiltration to tumors, inhibited the emergence of PD-1hi immune cells, indicating prevented T cell depletion. The elevated CD25 expression also supported the success in enhancing immune response reported by elevated T cell activity in spleen. Transcriptome analysis identified critical intracellular pathways regulated by the concurrent blockade of PD-L1 and VEGFR2. Conclusion: PD-L1 and VEGFR2- bispecific VH intracellular nanobody was highly biocompatible and showed the potential for combined anti-cancer therapy through long-term immune activation mediated by PD-L1/PD-1 checkpoint blockade and anti-metastasis mediated by VEGFR2 blockade.