Veana Therapeutics, Inc.

The pleiotropic cytokine, transforming growth factor (TGF)-β, and CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) play a critical role in actively suppressing antitumor immune responses. Evidence shows that TGF-β produced by tumor cells promotes tolerance via expansion of Tregs. Our group previously demonstrated that blockade of TGF-β signaling with a small molecule TGF-β receptor I antagonist (SM16) inhibited primary and metastatic tumor growth in a T cell dependent fashion. In the current study, we evaluated the effect of SM16 on Treg generation and function.

Using BALB/c, FoxP3eGFP and Rag^-/- mice, we performed FACS analysis to determine if SM16 blocked de novo TGF-β-induced Treg generation in vitro and in vivo. CD4⁺ T cells from lymph node and spleen were isolated from control mice or mice maintained on SM16 diet, and flow cytometry analysis was used to detect the frequency of CD4⁺CD25^-FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺ T cells. In vitro suppression assays were used to determine the ability to suppress naive T cell proliferation in vitro of both CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25^-FoxP3⁺ T cell sub-populations. We then examined whether SM16 diet exerted an inhibitory effect on primary tumor growth and correlated with changes in FoxP3⁺expression. ELISA analysis was used to measure IFN-γ levels after 72 h co-culture of CD4⁺CD25⁺ T cells from tumor-bearing mice on control or SM16 diet with CD4⁺CD25^- T cells from naive donors.

SM16 abrogates TGF-β-induced Treg generation in vitro but does not prevent global homeostatic expansion of CD4⁺ T cell sub-populations in vivo. Instead, SM16 treatment causes expansion of a population of CD4⁺CD25^-Foxp3⁺ Treg-like cells without significantly altering the overall frequency of Treg in lymphoreplete naive and tumor-bearing mice. Importantly, both the CD4⁺CD25^-Foxp3⁺ T cells and the CD4⁺CD25⁺Foxp3⁺ Tregs in mice receiving SM16 diet exhibited diminished ability to suppress naive T cell proliferation in vitro compared to Treg from mice on control diet.

These findings suggest that blockade of TGF-β signaling is a potentially useful strategy for blunting Treg function to enhance the anti-tumor response. Our data further suggest that the overall dampening of Treg function may involve the expansion of a quiescent Treg precursor population, which is CD4⁺CD25^-Foxp3⁺.

Tumors employ strategies to escape immune control. The principle aim of most cancer immunotherapies is to restore effective immune surveillance. Among the different processes regulating immune escape, tumor microenvironment-associated soluble factors, and/or cell surface-bound molecules are mostly responsible for dysfunctional activity of tumor-specific CD8⁺T cells. These dynamic immunosuppressive networks prevent tumor rejection at several levels, limiting also the success of immunotherapies. Nevertheless, the recent clinical development of immune checkpoint inhibitors or of molecules modulating cellular targets and immunosuppressive enzymes highlights the great potential of approaches based on the selective disruption of immunosuppressive networks. Currently, the administration of different categories of immunotherapy in combination regimens is the ultimate modality for impacting the survival of cancer patients. With the advent of immune checkpoint inhibitors, designed to mount an effective antitumor immune response, profound changes occurred in cancer immunotherapy: from a global stimulation of the immune system to a specific targeting of an immune component. This review will specifically highlight the players, the mechanisms limiting an efficient antitumor response and the current immunotherapy modalities tailored to target immune suppressive pathways. We also discuss the ongoing challenges encountered by these strategies and provide suggestions for circumventing hurdles to new immunotherapeutic approaches, including the use of relevant biomarkers in the optimization of immunotherapy regimens and the identification of patients who can benefit from defined immune-based approaches.