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

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. 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. 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. 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.