PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T-cell infiltration into pancreatic tumors

Abstract

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to late detection and resistance to conventional therapies. Published studies show that the PDA tumor microenvironment is predominantly infiltrated with immune suppressive cells and signals that if altered, would allow effective immunotherapy. However, single-agent checkpoint inhibitors including agents that alter immune suppressive signals in other human cancers such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and its ligand PD-L1, have failed to demonstrate objective responses when given as single agents to PDA patients. We recently reported that inhibition of the CTLA-4 pathway when given together with a T cell inducing vaccine gives objective responses in metastatic PDA patients. In this study, we evaluated blockade of the PD-1/PD-L1 pathway. We found that PD-L1 is weakly expressed at a low frequency in untreated human and murine PDAs but treatment with a granulocyte macrophage colony-stimulating factor secreting PDA vaccine (GVAX) significantly upregulates PD-L1 membranous expression after treatment of tumor-bearing mice. In addition, combination therapy with vaccine and PD-1 antibody blockade improved murine survival compared with PD-1 antibody monotherapy or GVAX therapy alone. Furthermore, PD-1 blockade increased effector CD8 T lymphocytes and tumor-specific interferon-γ production of CD8 T cells in the tumor microenvironment. Immunosuppressive pathways, including regulatory T cells and CTLA-4 expression on T cells were overcome by the addition of vaccine and low-dose cyclophosphamide to PD-1 blockade. Collectively, our study supports combining PD-1 or PD-L1 antibody therapy with a T cell inducing agent for PDA treatment.

Figure 1. Pancreatic cancer Cy/GVAX therapy upregulates pancreatic tumor expression of PD-L1 in human & murine pancreatic ductal adenocarcinoma (PDA)

(A) Representative H&E staining and IHC with αPD-L1 antibody on resected PDA from unvaccinated patients and patients who received the Cy/GVAX vaccine therapy two weeks prior to surgical resection. (B) Immunofluorescence staining with αPD-L1 antibody and FITC conjugated secondary antibody in liver tumors after Panc02 hemispleen injection comparing mice treated with Cy/GVAX on day 4 and 7 after tumor inoculation (w/GVAX) against mice not receiving GVAX therapy (w/o GVAX) (upper panel). Livers were harvested two weeks after tumor cell inoculation. DAPI staining of nuclei is shown in lower panel.

Figure 2. Combination therapy with Cy/GVAX and PD-1 or PD-L1 blockade improves clinical outcomes in a PDA mouse model

(A) Schema of tumor implantation by the hemispleen procedure and treatment with Cy, GVAX and αPD-1/αPD-L1 blockade as indicated. C57Bl/6 mice were challenged on day 0 with 2 x 10⁶ Panc02 tumor cells followed by administration of 100 mg/kg of Cy on day 3 and irradiated whole-cell vaccine on day 4, 7, 14 and 21. Anti-PD-1, anti-PD-L1 or IgG (5 mg/kg IP) were administered IP twice weekly until death starting on day 3. (B) Kaplan-Meier survival curves of mice that were implanted with PDA cells and were treated with different combinations of Cy, GVAX and the αPD-1 antibody. The percentages of mice that remained disease free at day 90 following tumor implantation and therapy with (C) Cy, GVAX and/or αPD-1 or (D) Cy, GVAX and αPD-L1 are shown. All the p values were yielded by comparing GVAX and/or αPD-1/αPD-L1 treatment groups with IgG treated group. (E) Kaplan-Meier survival curves of mice that were implanted with Panc02 cells via hemispleen technique and treated with different combinations of Cy, GVAX and αPD-L1 antibody. Data are represented as results obtained from experiments with 8-10 mice per group that were repeated at least twice. N.S. not significant.

Figure 3. Cy/GVAX combined with PD-1 blockade increases CD8⁺ T cells in PDAs

(A) Schema of immune analysis following tumor implantation by the hemispleen procedure and treatment with Cy (100 mg/kg) on day 3, GVAX on day 4, 7 and IgG/αPD-1/αPD-L1 (5 mg/kg IP) on day 3, 6, 10. Each experimental group consisted of five mice, pooled and analyzed individually in triplicates. Percentage of (B) CD8⁺ and (C) CD4⁺ tumor infiltrating lymphocytes among total lymphocytes in murine livers and total numbers of (D) CD8⁺ and (E) CD4⁺ tumor infiltrating lymphocytes after Panc02 hemispleen and indicated therapy. Data represent mean ± SEM from one representative experiment that was repeated at least twice. * p=0.04, TIL tumor-infiltrating lymphocytes.

Figure 4. Combinatorial treatment increases the percentage of IFNγ secreting CD8⁺ T cells and tumor specific CD8⁺ T cells in the tumor microenvironment

CD8⁺ T cells were isolated and purified from spleen and livers on day 13 after hemispleen implantation of Panc02 tumor cells. Tumor-bearing mice were treated with Cy, GVAX or αPD-1/αPD-L1 therapy as indicated. The percentage of IFNγ⁺ producing CD8⁺ T cells amongst all CD8⁺ T cells in (A) splenocytes and (B) tumor infiltrating lymphocytes is shown. ELISA assays were performed using autologous irradiated Panc02 tumor cells as antigenic targets for CD8⁺ T cells isolated from (C) spleen and (D) tumor infiltrating lymphocytes. Each experimental group consisted of five mice, pooled and analyzed individually in triplicates. Data represent mean ± SEM from one representative experiment that was repeated at least twice. *p<0.05, ** p<0,01, *** p< 0.001, TIL tumor-infiltrating lymphocytes

Figure 5. Cy/GVAX therapy with αPD-1 blockade overcomes immunosuppressive pathways

Following hemispleen implantation of Panc02 cells, tumor-bearing mice were treated with Cy (100 mg/kg) on day 3, GVAX on day 4, 7 and IgG/αPD-1/αPD-L1 (5 mg/kg IP) on day 3, 6, 10. Mice were sacrificed on day 13. (A) Fluorescence-activated cell sorting (FACS) density plot of CD4⁺CD25⁺Foxp3⁺ Tregs in TILs. (B) Histogram showing the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in CD4⁺ TILs. (C) Histogram showing the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in all TILs. (D) Histogram showing the total number of CD4⁺CD25⁺Foxp3⁺ Tregs infiltrating the tumors. (E) Representative FACS analysis of CTLA-4⁺CD8⁺ T cells in TILs. (F) Histogram showing the percentage of CTLA-4⁺CD8⁺ T cells within CD8⁺ T cells. (G) Histogram showing the percentage of CTLA-4⁺CD4⁺ T cells within CD4⁺ T cells. (H) Histogram showing the total number of CTLA-4⁺CD8⁺ TILs. (I) Histogram showing the total number of CTLA-4⁺CD4⁺ TILs. Each experimental group consisted of five mice, pooled and analyzed individually in triplicates. Data represent mean ± SEM from one representative experiment that was repeated at least twice. *p<0.05, **p<0.01, ***p<0.001. IgG, hamster IgG; TIL, tumor infiltrating lymphocytes