Journal: Biomaterials

Article Title: An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation.

doi: 10.1016/j.biomaterials.2022.121542

Figure Lengend Snippet: Fig. 2. Generation and characterization of a protein-based all-in-one cancer vaccine (DCpep6-4xE7ΔNLS-FlaB; DEF). (A) The vector maps and amino acid sequences of recombinant proteins are shown; in vivo DC-targeting peptide (DCpep6), tumor antigen (E7ΔNLS), and built-in flagellin adjuvant (FlaB). The E7ΔNLS tumor antigen was prepared by deleting the N-terminal nuclear localization sequence (NLS) of the HPV16 E7 protein. An all-in-one type TCV containing DCpep6, HPV E7ΔNLS and built-in FlaB adjuvant was constructed by recombinant DNA technology in the pET30a+ plasmid. 4xE7ΔNLS-FlaB (EF) was also generated as a non- CD11c+ cell targeting TCV. (B) Characterization of the recombinant proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and sub sequent Western blot analysis using mouse anti-FlaB or anti-E7 serum. Anti-FlaB or anti-E7 serum was induced by intraperitoneal immunization of FlaB or E7FL with complete Freund’s adjuvant. (C) Selection of optimal tumor antigen (E7ΔNLS) for the all-in-one cancer vaccine. C57BL/6 mice were implanted with TC-1 cells in the right midflank. When the tumor size reached approximately 3–5 mm in diameter, the tumor-bearing mice were subcutaneously vaccinated with 200 μl of PBS only (PBS; n = 5), 4 μg of FlaB (F; n = 5), 10 μg of E7 full length (E7FL; n = 5), 10 μg of E7FL plus 4 μg of FlaB (E7FL+F; n = 5), 8 μg of E7ΔNLS (E; n = 5), and 8 μg of E7ΔNLS plus 4 μg of FlaB (E+F; n = 5) in the peritumoral region three times at five-day intervals. The statistical significance of tumor suppression was calculated by two-way ANOVA. (D) Determination of TLR5-dependent NF-κB stimulating activity by FlaB, EF or DEF. The relative NF-κB activities were analyzed by using HEK- Blue™ hTLR5 cells and HEK-Blue™ detection assay systems. (E) Determination of dose-dependent cellular uptake of DEF in CD11c+ mouse BMDCs by FACS. BMDCs were incubated with various concentrations of EF or DEF for 2 h, and then intracellular localization of the recombinant proteins was determined by FACS analysis using anti-FlaB serum gating upon CD11c+ cell population. (F) Determination of the intracellular localization of DEF in BMDCs or RAW264.7 cells by confocal microscopic observation. BMDCs or RAW264.7 cells were incubated with 20 μg/ml of EF or DEF for 2 h, and intracellular localization of the recombinant proteins was determined by confocal microscopic observation. (G) Determination of energy-dependent cellular uptake of DEF in CD11c + cells. BMDCs were incubated with 20 μg/ml of DEF at 37 ◦C, 4 ◦C, or 37 ◦C in the presence of 0.1% NaN3, or 37 ◦C in the presence of 2 μg/ml cytochalasin B (CB) for 2 h, and then intracellular localization of the recombinant proteins was determined by FACS analysis gating upon CD11c+ cell population. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, nonsignificant.

Article Snippet: To test whether the EF or DEF proteins maintain TLR5-stimulating activities, we measured TLR5-dependent NF-κB-stimulating activity of the recombinant proteins by using HEK-BlueTM hTLR5 cells (InvivoGen, hκb-htlr-5) and HEK-BlueTM Detection (InvivoGen, hb-det2) assay systems following the manufacturer’s instructions.

Techniques: Plasmid Preparation, Recombinant, In Vivo, Adjuvant, Sequencing, Construct, Generated, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Selection, Activity Assay, Detection Assay, Incubation