Novel Protein-Based Vaccine against Self-Antigen Reduces the Formation of Sporadic Colon Adenomas in Mice

Abstract

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: (1) a selected tumor antigen; (2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and (3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

Keywords: Ascl2; T cell response; cancer vaccine; colon cancer; humoral response; mouse model.

Figure 1

KISIMA-Mash2 vaccine promotes T cell and humoral responses. C57BL/6 mice were treated with 6 administrations of KISIMA-Mash2 vaccine (Day 0, 14, 28, 58, 84 and 127). Thirteen days after the last vaccination, T cell immune response was assessed by ELISpot after stimulation of splenocytes with KISIMA-Mash2 vaccine overnight (A). Humoral response was determined in serum by detecting anti-Mash2 IgG by ELISA (B). *, p < 0.05; **, p < 0.01 (unpaired T test).

Figure 2

KISIMA-Mash2 vaccine is safe and promotes a humoral response in Apc^+/Min-FCCCmice. (A) Study design. Apc^+/Min-FCCC mice underwent colonoscopy (cross) prior to enrollment on study. Serum was collected (triangles) at 3 different time-points. Vaccine or vehicle control (arrows) was administered six times. All animals were necropsied one week after the last vaccine injection. (B) Average body weight of animals obtained weekly for the duration of the study. (C) Humoral response determined in serum at Week 17 by detecting anti-Mash2 IgG by ELISA.

Figure 3

Combination of KISIMA-Mash2 vaccine and anti-PD-1 treatment is safe and promotes T cell immunity in Apc^+/Min-FCCC mice. (A) Study design. Animals underwent colonoscopy (cross) prior to enrollment on study. Serum was collected (triangles) at baseline and at three additional intervals throughout the study. Vaccine or vehicle control (arrows) was administered six times, and anti-PD1 or buffer control (stars) was administered in three cycles of three-weekly injections. All animals were necropsied one week after the last vaccine injection. (B) Average body weight of animals obtained weekly for the duration of the study. (C) T cell immune response assessed by ELISpot on ex vivo splenocytes after overnight stimulation with overlapping peptide pools derived from Mash2 antigen. Bar represents the mean of each group for each peptide pool stimulation. ** p < 0.01; *** p < 0.001; **** p < 0.0001 (2-way ANOVA followed by Tukey’s multiple comparison) (D) Humoral response determined in serum at Week 3, Week 9, and Week 17 by detecting anti-KISIMA-Mash2 IgG by ELISA. Line represents the mean for each group. p < 0.05 between Vaccinated vs. Control or Anti-PD-1 groups and p < 0.001 between Combo vs. Control or Anti-PD-1 groups at Week 17 (Two-way ANOVA followed by Tukey’s multiple comparison).

Figure 3

Combination of KISIMA-Mash2 vaccine and anti-PD-1 treatment is safe and promotes T cell immunity in Apc^+/Min-FCCC mice. (A) Study design. Animals underwent colonoscopy (cross) prior to enrollment on study. Serum was collected (triangles) at baseline and at three additional intervals throughout the study. Vaccine or vehicle control (arrows) was administered six times, and anti-PD1 or buffer control (stars) was administered in three cycles of three-weekly injections. All animals were necropsied one week after the last vaccine injection. (B) Average body weight of animals obtained weekly for the duration of the study. (C) T cell immune response assessed by ELISpot on ex vivo splenocytes after overnight stimulation with overlapping peptide pools derived from Mash2 antigen. Bar represents the mean of each group for each peptide pool stimulation. ** p < 0.01; *** p < 0.001; **** p < 0.0001 (2-way ANOVA followed by Tukey’s multiple comparison) (D) Humoral response determined in serum at Week 3, Week 9, and Week 17 by detecting anti-KISIMA-Mash2 IgG by ELISA. Line represents the mean for each group. p < 0.05 between Vaccinated vs. Control or Anti-PD-1 groups and p < 0.001 between Combo vs. Control or Anti-PD-1 groups at Week 17 (Two-way ANOVA followed by Tukey’s multiple comparison).

Figure 4

Efficacy of KISIMA-Mash2 vaccine and anti-PD-1 combination treatment on tumor development in Apc^+/Min-FCCC mice. (A) Number of colon adenomas per mouse in animals that were tumor-free at treatment initiation (after removal of outliers). (B) Number of colon microadenomas per mouse in animals that were tumor-free at treatment initiation after removal of outliers. *, p > 0.05. All other statistical comparisons were not significant.

Figure 5

Analysis of CD3+ cells infiltration in Apc^+/Min-FCCC mice. Representative colon adenomas, stained with hematoxylin and eosin (H&E, left panel) and anti-CD3 antibody (right panel), from mice treated with buffer (Buffer), anti-PD1 alone (anti-PD1), KISIMA-Mash2 vaccine alone (Vaccine), or the KISIMA-Mash2 vaccine and anti-PD-1 (Combination). Boxes contain a high power view (200×) of the 40× areas positive for CD3 staining. Representative intraepithelial lymphocytes are highlighted with arrows on the 200× images.

Figure 6

Combination of KISIMA-Mash2 vaccine and anti-PD-1 treatment results in higher T cell infiltration in Apc^+/Min-FCCC mice. Anti-CD3 staining of adenomas from mice treated with buffer (Buffer), anti-PD1 alone ‘anti-PD1), KISIMA-Mash2 vaccine alone (Vaccine), or the KISIMA-Mash2 vaccine and anti-PD-1 combination (Combination). Box plots indicate the number of intraepithelial CD3+ lymphocytes per area (mm²) of dysplasia in each animal. Kruskal-Wallis test comparing all 4 groups simultaneously p = 0.002. p values from two-sided Wilcoxon tests are shown on the graph.