Fig. 3
Efficacy of FAP1V2 transient expression in mediating PD-1 checkpoint blockade immune inhibition of LLC progress in mice models. A Schematic diagram and (B) flow cytometry assay showed that the blockage of cell surficial PD-L1 by FAP1V2 or FAP1 caused reduced activity of PD-L1 to bind commercial PE-labeled human PD-1. The cells were transfected with pEGFP-C1, pEGFP-C1-AP1 or pEGFP-C1-AP1V2, respectively. The cells were not treated with 0.1% Triton before cell cytometry analysis. C to (F) Cell flow cytometry and antibody competition assays demonstrated that intracellular antibodies FAP1 and FAP1V2, or FAV2 and FAP1V2 could selectively bind to PD-L1 or VEGFR2 expressed by LLC cells. Initially, 293 T cells were separately transfected with pEGFP-C1-AP1, pEGFP-AV2 or pEGFP-C1-AP1V2. Following co-incubation of their lysates with LLC cells (fixed with paraformaldehyde), competitive binding assays were performed using commercial anti-mouse PD-L1 and VEGFR2 antibodies (conjugated with APC) against the corresponding antigens. Panel (C) shows LLC cells in four conditions, from left to right: Control: No co-incubation with 293 T lysates or commercial antibody. Anti-PD-L1 antibody: Co-incubated with commercial anti-mouse PD-L1 antibody. Single-target intrabody (FAP1) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing single-target intrabody FAP1, followed by addition of commercial anti-mouse PD-L1 antibody. Double-target intrabody (FAP1V2) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing double-target intrabody FAP1V2, followed by addition of commercial anti-mouse PD-L1 antibody. D Column graph illustrates the average fluorescence values of results shown in (C). Panel (E) shows LLC cells in four conditions, from left to right: Control: No co-incubation with 293 T lysates or commercial antibody. Anti-VEGFR2 antibody: Co-incubated with commercial anti-mouse VEGFR2 antibody. Single-target intrabody (FAV2) plus anti-PD-L1 antibody: Co-incubated with 293 T lysate expressing single-target intrabody FAV2, followed by addition of commercial anti-mouse VEGFR2 antibody. Double-target intrabody (FAP1V2) plus anti-VEGFR2 antibody: Co-incubated with 293 T lysate expressing double-target intrabody FAP1V2, followed by addition of commercial anti-mouse VEGFR2 antibody. F Column graph presents the average fluorescence values of results shown in (E). This experiment was independently repeated three times. Based on three mean fluorescence values (MFV) of the cell populations from each sample, as reported by flow cytometer (Channel APC-A), the data for the mean fluorescence values detected at Channel APC-A are presented as Mean MFV ± SEM. *, **, *** represent significant differences compared to the control group at the levels of P < 0.05, P < 0.01, P < 0.001, respectively. #, ##, ### indicate significant differences between treatment groups at the levels of P < 0.05, P < 0.01, P < 0.001, respectively. G to (I) The effect of anti-PD-L1-anti-VEGFR2 chimeric intrabody FAP1V2 on enhancing immune activity of suppressing tumorigenesis and growth of LLC cells in C57BL/6 mice. G Timeline for studying transient FAP1V2 expression mediated anticancer therapy via immune activation and antimetastatic effects. H Tumor size varied in the mice injected with LLC cancer cells which transiently expressed FAP1, FAV2 or FAP1V2 intrabody, respectively. The group of mice inoculated with LLC cells transiently transfected with blank plasmid (pEGFP-C1) were set as a control. The LLC cells were respectively subcutaneously injected to the right armpits first and then to the left armpits 4 days later, which can maintain the transient expression of the intrabodies for total 6–8 days in the beginning. The data on tumor sizes for each group, which included tumors located in the same side of armpits from 6 mice, are presented as the Mean Tumor size ± SEM. (I) Images of the tumors separated from the right and left armpits of the mice which were sacrificed at Day 28