The recent clinical successes of CTLA4 and PD-1 blockade have clearly demonstrated the therapeutic efficacy to enhance anticancer immune responses. Novel targets of immunomodulation are now under investigation to increase the number of immunotherapeutic options available for relapsing/refractory patients. Activation of co-stimulatory molecules with agonist monoclonal antibody (mAbs) represents the most rationale alternative option. Based on the impressive preclinical activity of stimulating the glucocorticoid-induced TNF receptor (GITR) with the mAb DTA-1, an agonist anti-human GITR mAb has very recently entered the clinical evaluation. Although DTA-1, as a monotherapy, proved effective in curing established immunogenic tumors, it failed against advanced poorly immunogenic models, such as B16 melanoma. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation with the agonist antibody DTA-1 affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition, and verified their functional changes in vitro.

In vivo, the comparison of advanced versus early B16 microenvironment before treatment revealed a significant increase in Treg frequency, which caused the Teff/Treg ratio to decrease, and a dysfunctional Teff phenotype. Moreover, B16 cells, in the setting of established tumors, expressed lower levels of MHC-I and MHC-II molecules. DTA-1 consistently reduced intratumor Treg frequency and expression of suppression markers in both curative and refractory conditions. However, Teff activation and tumor-associated MHCs were induced only when DTA-1 was administered in curative conditions.

In vitro, pre-incubation of Tregs with DTA-1 completely abolished their ability to inhibit proliferation of target Teff, without affecting Treg expression of the lineage markers Foxp3 and Helios. In addition, treatment with DTA-1, in comparison with the matched IgG isotype control, boosted the release of IFN-gamma, TNF-alpha, IL-17 and IL-6 cytokines in cultures of Tregs, pointing to the promotion of a Th1/Th17 differentiation profile following GITR engagement. Interestingly, when B16 cells were added in culture with Tregs and incubated with DTA-1, they up-regulated MHC-I and MHC-II molecules in a Treg and/or DTA-1 dependent manner. The presence of tumor cells during exposure of Tregs to anti-GITR stimulation did not further alter their function and phenotype, indicating a major direct effect of DTA-1 on Tregs. To confirm the functional modulation of GITR stimulation in human Tregs, we tested their activity in vitro upon pre-incubation with a recombinant human GITR ligand (GITRL). We consistently found that proliferation of activated Teff was no longer suppressed if Tregs were previously treated with GITRL. These results validate GITR as a target to revert immune tolerance in the human setting.

Our data indicate that anti-GITR therapy counteracts Treg-mediated immunosuppression independently from the stage of the disease, whereas T-cell activation and tumor immune recognition are favored selectively in the presence of low tumor burden. These findings suggest that, in the setting of advanced and poorly immunogenic tumors, anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing.

Citation Format: Roberta Zappasodi, Sadna Budhu, Cailian Liu, Kyle Draleau, David Schaer, Hong Zhong, Xia Yang, Taha Merghoub, Jedd D. Wolchok. Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B183.