Combination therapy targeting both innate and adaptive immunity improves survival in a pre-clinical model of ovarian cancer

Abstract

Background: Despite major advancements in immunotherapy among a number of solid tumors, response rates among ovarian cancer patients remain modest. Standard treatment for ovarian cancer is still surgery followed by taxane- and platinum-based chemotherapy. Thus, there is an urgent need to develop novel treatment options for clinical translation.

Methods: Our approach was to analyze the effects of standard chemotherapy in the tumor microenvironment of mice harboring orthotopic, syngeneic ID8-Vegf-Defb29 ovarian tumors in order to mechanistically determine a complementary immunotherapy combination. Specifically, we interrogated the molecular and cellular consequences of chemotherapy by analyzing gene expression and flow cytometry data.

Results: These data show that there is an immunosuppressive shift in the myeloid compartment, with increased expression of IL-10 and ARG1, but no activation of CD3⁺ T cells shortly after chemotherapy treatment. We therefore selected immunotherapies that target both the innate and adaptive arms of the immune system. Survival studies revealed that standard chemotherapy was complemented most effectively by a combination of anti-IL-10, 2'3'-cGAMP, and anti-PD-L1. Immunotherapy dramatically decreased the immunosuppressive myeloid population while chemotherapy effectively activated dendritic cells. Together, combination treatment increased the number of activated T and dendritic cells as well as expression of cytotoxic factors. It was also determined that the immunotherapy had to be administered concurrently with the chemotherapy to reverse the acute immunosuppression caused by chemotherapy. Mechanistic studies revealed that antitumor immunity in this context was driven by CD4⁺ T cells, which acquired a highly activated phenotype. Our data suggest that these CD4⁺ T cells can kill cancer cells directly via granzyme B-mediated cytotoxicity. Finally, we showed that this combination therapy is also effective at delaying tumor growth substantially in an aggressive model of lung cancer, which is also treated clinically with taxane- and platinum-based chemotherapy.

Conclusions: This work highlights the importance of CD4⁺ T cells in tumor immunology. Furthermore, the data support the initiation of clinical trials in ovarian cancer that target both innate and adaptive immunity, with a focus on optimizing dosing schedules.

Keywords: CD4+ T cells; Cancer immunotherapy; Combination therapy; Innate immunity; Ovarian cancer.

Fig. 1

Treatment with paclitaxel and carboplatin induces acute immunosuppression that is mediated by innate immune cells. Mice were inoculated orthotopically with ID8-Vegf-Defb29 ovarian cancer cells. Eight days later, the mice were injected with vehicle (Veh) or chemotherapy (Chemo). Two days later, peritoneal cells were harvested for analysis. a Volcano plots of gene expression data sets derived from FACS-sorted leukocytes (CD11b⁺ and CD3⁺). All probe sets are shown. The top differentially expressed genes in the myeloid population are named, and highlight coloring was applied to significantly differentially expressed (adj. p-value < 0.05) probe sets. The experiment was performed once with n = 3 biological replicates. b A heatmap of the top 35 upregulated genes after chemotherapy treatment in FACS-sorted CD11b⁺ cells. c Peritoneal cell suspensions were assessed by flow cytometry. Bar graphs show quantification of flow cytometry gating of CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells. d Flow cytometry gating of subsets of MHCII⁺ mature dendritic cells are shown as scatter plots and quantified at right. e Flow cytometry gating of subsets of F4/80⁺ macrophages are shown as scatter plots and quantified at right. Increased numbers of immunosuppressive ARG1⁺ IL-10⁺ myeloid cells are observed following chemotherapy. The experiment was performed twice with n = 4 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001

Fig. 2

STING agonism combined with neutralization of IL-10 and PD-L1 after chemotherapy increases survival. Different combinations of chemotherapy and immunotherapy were tested in vivo for synergy. Kaplan-Meier curves are shown for mice treated with chemotherapy and (a) anti-IL-10, GR-MD-02, and anti-4-1BB, b 2′3’-cGAMP, GR-MD-02, and anti-4-1BB (c) gemcitabine, 2′3’-cGAMP, and anti-PD-L1, d anti-IL-10, 2′3’-cGAMP, and anti-4-1BB, or (e) anti-IL-10, 2′3’-cGAMP, and anti-PD-L1. a-e All combination treatments were compared to chemotherapy and isotype control for immunotherapy (Chemo) 8 days after inoculation of ID8-Vegf-Defb29 cells. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed with biological replicates twice. Statistics were calculated relative to the group treated with chemotherapy only using the Log-rank (Mantel-Cox) test. ** p ≤ 0.01, **** p ≤ 0.0001

Fig. 3

Combination therapy reverses the myeloid cell-mediated immunosuppression and promotes infiltration of activated DCs and T cells. a Peritoneal cell suspensions from tumor-bearing mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chemo and IT (Combo) were assessed by flow cytometry 4 days after initiation of treatment. a, b Decreased numbers of myeloid cells with immunosuppressive phenotypes are observed upon Combo treatment. a Decreased numbers of F4/80⁺ macrophages are observed upon treatment with immunotherapy (IT and Combo) (b) Flow cytometry gating of subsets of ARG1⁺IL-10⁺ myeloid cells are shown as scatter plots and quantified at right. c, d Increased numbers of mature dendritic cells are observed upon Combo treatment. c Flow cytometry gating of subsets of CD11c⁺ dendritic cells are shown as scatter plots and quantified at right. Numbers of CD11c⁺ cells expressing co-stimulatory molecules are quantified. d STING activation is pharmacodynamically confirmed by increased median fluorescence intensity of IRF3. e The adaptive immune system is also impacted by Combo therapy. Flow cytometry gating of subsets of CD3⁺ T cells are shown as scatter plots and quantified at right. Increased numbers of CD4⁺ T cells expressing the activation marker CD69, the cytolytic molecule CD107a, and the pro-inflammatory cytokine IL-2 are observed. Increased numbers of CD8⁺ T cells expressing the cytolytic molecule GZMB are shown. The experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 4

Survival benefit conferred by the combination therapy is superior to chemotherapy or immunotherapy alone and strongly influenced by dosing schedule. Different dosing schedules were tested to understand the temporal interaction between chemotherapy and immunotherapy in ID8-Vegf-Defb29-tumor bearing mice. Each is depicted above the Kaplan-Meier curves. a A Kaplan-Meier curve is shown comparing combination therapy (Combo) to chemotherapy (Chemo) or immunotherapy (IT) alone as well as vehicle only (Vehicle). b A Kaplan-Meier curve is shown comparing 3 weeks of treatment (Combo) to 1 week of immunotherapy treatment (Combo short) following chemotherapy. c A Kaplan-Meier curve is shown comparing immunotherapy initiated on the same day as chemotherapy (Combo) to immunotherapy initiated 4 days later (Delayed IT). d A Kaplan-Meier curve is shown comparing combination therapy initiated on day 8 (Combo) to combination therapy initiated on day 22 (Combo late). b-d Treatment groups are compared to chemotherapy and isotype control (Chemo). The number of mice per group (n) and median survival (ms) are listed. All experiments were performed with biological replicates at least twice. Statistics were calculated using the Log-rank (Mantel-Cox) test. *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 5

CD4⁺ T cells are critical for the efficacy of the combination therapy. a Specific immune cell subsets (CD4⁺ T cells, CD8⁺ T cells, or CD11b⁺ cells) were depleted to explore their relative contribution to the observed efficacy. Kaplan-Meier curves are shown for all groups described compared to isotype control. The number of mice per group (n) and median survival (ms) are listed. All experiments were performed twice with n = 5 biological replicates. Dosing schedule is shown at the top of the figure. Statistics were calculated using the Log-rank (Mantel-Cox) test. ** p ≤ 0.01, **** p ≤ 0.0001. b-f Peritoneal cell suspensions from tumor-bearing mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chemo and IT (Combo) were assessed by flow cytometry 13 days after initiation of treatment. (b) Bar graphs show quantification of flow cytometry gating of CD4⁺ and CD8⁺ T cells. (c) Increased numbers of RORγt- and FoxP3-expressing CD4⁺ T cells are observed with Combo therapy. (d) CD4⁺ T cells expressing activation markers are observed. (e) Increased numbers of dendritic cells are observed upon Combo treatment even at this late timepoint. (f) Flow cytometry gating of subsets of GZMB expressing CD4⁺ T cells are shown as scatter plots and quantified at right. MHCII-expression on cancer cells is confirmed. The experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 6

Efficacy of combination therapy is similarly observed in a subcutaneous lung cancer model. Combination therapy was tested in the murine LLC lung cancer model. Tumors were allowed to grow to an average of 100mm³ per group before initiation of treatment (red arrow). Average fold change of tumor volume of mice treated with combination therapy (Combo), chemotherapy (Chemo) alone, or immunotherapy (IT) alone as well as vehicle only (Vehicle). The number of mice per group (n) are listed. All experiments were performed with biological replicates twice. Statistics were calculated using two-way ANOVA and the Log-rank (Mantel-Cox) test. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001