Therapeutic PD-1 Pathway Blockade Augments with other Modalities of Immunotherapy to Prevent Immune Decline in Ovarian Cancer

The tumor microenvironment mediates induction of the immunosuppressive programmed death-1 (PD-1) pathway, targeted interventions against which can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1) expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TILs) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including macrophages (TAM), dendritic cells (DC) and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing GM-CSF or FLT3 ligand) and co-stimulation by agonistic α 4-1BB or TLR 9 ligand, antibody mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8 + T cells, inhibition of suppressive T regulatory cells (Tregs) and MDSC, upregulation of effector T cell signaling molecules and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control. mechanisms for the accumulation of these immunosuppresive myeloid cells or Tregs. In this study, we demonstrate a cross-regulation among these cell types using the ID8 syngeneic mouse model of epithelial ovarian cancer. We provide evidence that T cell dysfunction can be reversed by targeting the PD-1 pathway simultaneously in all these cell types. We found that expansion of ovarian antigen-specific CD8 + TILs was dependent on the amount of PD-L1 signaling by tumor cells, tumor-derived myeloid cells and Tregs. Furthermore, combining PD-1 blockade with a single dose of GVAX or FVAX vaccination resulted in enhanced clonal expansion of antigen-specific CD8 + T cells and tumor control. Finally, we observed a further boost of CD8 + T cell function when PD-L1 expressed by tumor cells versus tumor stroma or infiltrating leukocytes. Future studies using PD-L1 KO mice and bone marrow chimeras will be required to clarify this point. PD-L1 blockade overcame the upregulation of MDSCs and pDCs by GVAX and FVAX, respectively. Previous studies reported that FVAX was more effective in combination with blockade therapy than GVAX (35). Our study used different clones of rat anti-mouse PD-1 (RMP1-30), and rat anti-mouse PD-L1 (10F.9G2) compared to Bioxcell antibodies, α PD-1 (RMP1-14), α PD-L1 (9G2) used in their study which may have accounted for eliminating the differences previously observed between the two vaccine preparations. Our findings have important implications in understanding the immunobiology of ovarian cancer and developing rational therapies. We have previously described the classification of ovarian cancers in tumors with and tumors without intraepithelial TILs (4). The present findings suggest that tumors lacing intraepithelial TILs may have a very active PD-L1 mediated suppression, leading to depletion of TILs. This would be in agreement with observations by Hamanishi et al . reporting that increased expression of PD-L1 (but not PD-L2) is associated with poor survival in patients with advanced ovarian cancer, similar to the lack of intraepithelial TILs (10). Our finding of high frequency of Treg cells in tumors lacking TILs is consistent with an association of Treg cells with poor outcome in human ovarian cancer (4) and with the observation that in these tumors we found increased expression of vascular endothelial growth factor (4), which also correlates with Treg cell preponderance in the tumor microenvironment (34). Based on the present findings, human ovarian cancer lacking intraepithelial TILs should not lack intrinsic immunogenicity, consistent with the notion that most advanced ovarian cancers express known tumor-associated antigens and harbor tons of non-synonymous mutations, which could give rise to immunogenic epitopes. Rather, powerful immunosuppressive factors would eliminate TILs, suggesting that reversal of these factors would restore immune recognition and attack. PD-L1 blockade could be an important step towards reversing such immunosuppressive environment, but additional maneuvers are likely required to optimize tumor immune attack. In the present of a whole tumor antigen enhanced the ability of PD-L1 blockade. in human patients with ovarian cancer treated with GVAX 55), vaccine was ineffective in the mouse, quite beneficial in association with checkpoint significant activation tumor microenvironment homeostatic increased MDSC pDCs no Treg