Agonist anti-GITR antibody significantly enhances the therapeutic efficacy of Listeria monocytogenes-based immunotherapy

Abstract

Background: We previously demonstrated that in addition to generating an antigen-specific immune response, Listeria monocytogenes (Lm)-based immunotherapy significantly reduces the ratio of regulatory T cells (Tregs)/CD4⁺ and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Since Lm-based immunotherapy is able to inhibit the immune suppressive environment, we hypothesized that combining this treatment with agonist antibody to a co-stimulatory receptor that would further boost the effector arm of immunity will result in significant improvement of anti-tumor efficacy of treatment.

Methods: Here we tested the immune and therapeutic efficacy of Listeria-based immunotherapy combination with agonist antibody to glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in TC-1 mouse tumor model. We evaluated the potency of combination on tumor growth and survival of treated animals and profiled tumor microenvironment for effector and suppressor cell populations.

Results: We demonstrate that combination of Listeria-based immunotherapy with agonist antibody to GITR synergizes to improve immune and therapeutic efficacy of treatment in a mouse tumor model. We show that this combinational treatment leads to significant inhibition of tumor-growth, prolongs survival and leads to complete regression of established tumors in 60% of treated animals. We determined that this therapeutic benefit of combinational treatment is due to a significant increase in tumor infiltrating effector CD4⁺ and CD8⁺ T cells along with a decrease of inhibitory cells.

Conclusion: To our knowledge, this is the first study that exploits Lm-based immunotherapy combined with agonist anti-GITR antibody as a potent treatment strategy that simultaneously targets both the effector and suppressor arms of the immune system, leading to significantly improved anti-tumor efficacy. We believe that our findings depicted in this manuscript provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.

Keywords: Anti-GITR antibody; Co-stimulation; Immune tolerance; Immunotherapy; Listeria vaccine; Lm-LLO-E7.

Fig. 1

a. Schematic of experiment. Starting on day 12 of tumor growth, TC-1 tumor-bearing, 6–8-week-old C57BL/6 female mice (n = 10 per group) were given anti-GITR Ab (i.p., 5 mg/kg daily, total 4 doses) along with Listeria-based E7 vaccine (i.p., two doses) every 7 days. Tumor growth and survival were measured until the end of the experiment. b. Tumor growth for individual mice in each group is presented. The number of mice with completely regressed tumors out of the 10 mice in the group is indicated. c. Averaged tumor volumes following various treatments. Statistical significance is shown for day 24. d. Kaplan-Meier plot for survival. e. SK Plot showing the tumor volume and survival for each mouse at different days. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001)

Fig. 2

Six days after the second Listeria-based immunotherapy treatment, mice were euthanized and tumors harvested and profiled for a. total number of CD4⁺ T cells per 1e⁶ tumor cells; b. total number of Tregs (CD4⁺FoxP3⁺) per 1e⁶ tumor cells; c total number of non-Tregs (CD4⁺FoxP3⁻) per 1e⁶ tumor cells; and d. Percentage of Tregs within the CD4⁺ T cell population. (*P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001). Experiment was repeated twice with n = 5/group with similar results

Fig. 3

Six days after the second Listeria-based immunotherapy treatment, mice were euthanized and tumors harvested and profiled for a. total number of CD8⁺ T cells; and b. total number of E7-specific CD8⁺ T cells (CD8⁺E7⁺) per 1e⁶ tumor cells. Ratios for CD8⁺/Tregs (c) and CD8⁺E7⁺/Tregs (d) were calculated. e. Six days after the second treatment with Listeria-based immunotherapy treatment, mice were euthanized and spleens were collected for ELISPOT analysis. Antigen-specific CD8⁺ T cells in the presence of E7_49–57 peptide versus irrelevant peptide control was assayed by ELISPOT. Values are presented as number of spots from E7_49–57 restimulated culture minus irrelevant Ag re-stimulated culture per million splenocytes. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). Experiment was repeated twice with n = 5/group with similar results

Fig. 4

Six days after the second treatment with Listeria-based immunotherapy treatment, mice were euthanized and tumors harvested and profiled for a. total number of GR1⁺CD11b⁺ MDSCs per 1e⁶ tumor cells. Ratios for CD8⁺/MDSCs (b) and CD8⁺E7⁺/MDSCs (c) were calculated. (*P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001). Experiment was repeated twice with n = 5/group with similar results