TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner

Abstract

Our neoadjuvant clinical trial of a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) revealed the development of tertiary lymphoid aggregates (TLAs) within the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment 2 weeks after GVAX treatment. Microarray studies revealed that multiple components of the TGF-β pathway were suppressed in TLAs from patients who survived greater than 3 years and who demonstrated vaccine-enhanced mesothelin-specific T cell responses. We tested the hypothesis that combining GVAX with TGF-β inhibitors will improve the anti-tumor immune response of vaccine therapy. In a metastatic murine model of pancreatic cancer, combination therapy with GVAX vaccine and a TGF-β blocking antibody improved the cure rate of PDA-bearing mice. TGF-β blockade in combination with GVAX significantly increased the infiltration of effector CD8+ T lymphocytes, specifically anti-tumor-specific IFN-g producing CD8+ T cells, when compared to monotherapy controls (all p < 0.05). TGF-β blockade alone did not deplete T regulatory cells (Tregs), but when give in combination with GVAX, GVAX induced intratumoral Tregs were depleted. Therefore, our PDA preclinical model demonstrates a survival advantage in mice treated with an anti-TGF-β antibody combined with GVAX therapy and provides strong rational for testing this combinational therapy in clinical trials.

Keywords: TGF-beta; immunotherapy; pancreatic cancer; regulatory T cells; vaccine.

Figure 1. Combination therapy with GVAX and αTGF-β blockade improves clinical outcomes in a PDA mouse model

A. Schema of tumor implantation by the hemispleen procedure and treatment with GVAX and αTGF-β blockade as indicated. C57Bl/6 mice were challenged on day 0 with 2 × 10⁶ Panc02 tumor cells followed by administration of irradiated whole cell GM-CSF Panc02 GVAX on days 4, 11, 18. αTGF-β or IgG was administered IP at 100 ug three times weekly for 3 weeks starting on day 3. B. The percentage of mice that remained disease free at day 90 following Panc02 tumor implantation with GVAX and/or αTGF-β/IgG. C. Schema of an additional PDA tumor implantation model consisting of 1 × 10⁵ KPC tumor cells injected on day 0 via hemispleen technique in C57Bl/6 mice. A single dose of irradiated GVAX was administered on day 4. αTGF-β or IgG was administered on days 3, 5 and 7 at 100 ug IP. D. The percentage of mice remaining disease free at day 90 following KPC tumor implantation with GVAX and/or αTGF-β/IgG. E. Schema of tumor implantation by the hemispleen procedure and treatment with GVAX, TGF-β blockade and αPD-1 as indicated. C57Bl/6 mice were challenged on day 0 with 2 × 10⁶ Panc02 tumor cells followed by administration of irradiated whole cell GM-CSF Panc02 GVAX on days 4, 11 and 18. αTGF-β or IgG was administered IP at 100 ug three times weekly for 3 weeks starting on day 3. αPD-1 was administered IP at 100 ug twice weekly for 3 weeks starting on day 3. F. The percentage of mice that remained disease free at day 90 following Panc02 tumor implantation with GVAX and/or αTGF-β/IgG and/or αPD-1. G. Kaplan-Meier survival curves of mice that were implanted with Panc02 tumor cells via hemispleen technique and treated with different combinations of Panc02 GVAX, αTGF-β, IgG and/or αPD-1. Data are represented as results obtained from experiments with 8 to 10 mice per group, pooled and repeated at least twice. NS, not significant; *p < 0.05. PDA, pancreatic ductal adenocarcinoma. IP, intraperitoneal.

Figure 2. Combination therapy with GVAX and αTGF-β decreases CD4⁺ T cell presence including Tregs in PDA

A. Schema of immune analysis following tumor implantation by the hemispleen procedure and treatment with αTGF-β or IgG (100 ug IP) on days 3, 5, 7 and GVAX on day 4. B. The percentage of CD4⁺ T cells among total lymphocytes and C. the total number of CD4⁺ TILs after Panc02 hemispleen and the indicated therapy. D. Fluorescence-activated cell sorting (FACS) cytometry gating schema and density plot for CD4⁺CD25⁺Foxp3⁺ Tregs among TILs. Histogram showing E. the percentage of Tregs amongst TILs and F. the total number of Tregs in the PDA TME after the indicated therapy. Each experiment consisted of 3 or 6 mice per group, pooled and analyzed individually in duplicate. Data represent mean ± SEM from one experiment repeated at least twice. *p < 0.05, ^**p < 0.01. TILs, tumor infiltrating lymphocytes. PDA, pancreatic ductal adenocarcinoma. TME, tumor microenvironment. Tregs, regulatory T cells. IP, intraperitoneal

Figure 3. Combination therapy enhances the population of IFNγ⁺ producing CD8⁺ T cell infiltration in the TME in the setting of decreased Tregs

A. The percentage of CD8⁺ T cell among TILs and B. total number of CD8⁺ T cells within the TME. C. The percentage of IFNγ⁺ producing CD8⁺ T cells among all CD8⁺ T cells in the TME. The ratio of D. CD8⁺ TIL to Tregs and E. CD8⁺IFNγ⁺ T cells to Tregs. Each experiment consisted of 3 or 6 mice per group, pooled and analyzed individually in duplicate. Data represent mean ± SEM from one experiment repeated at least twice. *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. TILs, tumor infiltrating lymphocytes. TME, tumor microenvironment. Tregs, regulatory T cells

Figure 4. Tumor specific CD8⁺ T cells in the TME is enhanced with combinatorial immunotherapy

Irradiated Panc02 tumor cells were used as antigenic targets for CD8⁺ T cells isolated from A. TILs and B. the spleen. Each experiment consisted of 3 or 6 mice per group, pooled and analyzed individually in duplicate. Data represent mean ± SEM from one experiment repeated at least twice. *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. TILs, tumor infiltrating lymphocytes. TME, tumor microenvironment

Figure 5. A diagram illustrating the potential effects of TGF-β inhibitors in combination with the pancreatic cancer vaccine therapy