PROJECT SUMMARY Although immune checkpoint blockade (ICB) has become a standard cancer therapy, most patients – including ~70% of those with non-small cell lung cancer (NSCLC) – fail to respond to ICB. Accumulation of transforming growth factor-β (TGFβ) in the tumor microenvironment (TME) can drive immune dysfunction by inducing regulatory T cells (Tregs) and inhibiting effector CD8+ T cells, resulting in ICB resistance. However, given the pleotropic, multifunctional nature of TGFβ, systemic inhibition has been clinically challenging with multiple side effects. Thus, developing translatable approaches to inhibit TGFβ within the TME is of paramount importance. Glycoprotein-A repetitions predominant (GARP), encoded by LRRC32, is a cell surface, non-signaling, docking, and activating receptor for latent TGFβ that is highly expressed in cancer cells (including lung cancer), activated Tregs, and platelets. We discovered that genetic deletion of Lrrc32 in Tregs or platelets enhanced protection against mouse models of inflammation-associated colorectal cancer and colon cancer. We validated GARP as a therapeutic target by generating a monoclonal antibody against it (called PIIO-1). PIIO-1 diminishes TGFβ signaling in the TME specifically due to its selective ability to recognize GARP on Tregs but not platelets. To test the translational capacity of PIIO-1, we generated a human LRRC32 knock-in mouse that globally replaces the murine extracellular components of GARP with the human sequence – this allows us to study safety and efficacy of PIIO-1 in murine models. We found that PIIO-1 has single agent activity against multiple cancer models and can promote CD8+ T cell function in vivo, as evidenced by reduced CD8+ T cell exhaustion and increased CXCR3+ CD8+ T effector function in the TME. Prolonged exposure to PIIO-1 did not induce platelet-related toxicity. Finally, we found that PIIO-1 improves therapeutic efficacy of PD-1 blockade against murine models of lung cancer. Given these exciting discoveries, we hypothesize that disrupting the GARP-TGFβ axis within the TME using our anti-GARP antibody PIIO-1 will augment the anti-tumor immunity of CD8+ T cells and overcome ICB resistance in patients with NSCLC. We will test this hypothesis with the following specific aims: Aim 1. Define how GARP inhibits CD8+ T cell differentiation programming and effector function in the TME. Aim 2. Evaluate and characterize PIIO-1 as an immunotherapeutic agent against preclinical models of NSCLC, alone or in combination with ICB, to support the development and initiation of a potentially best-in-class anti-GARP antibody phase I clinical trial. This study will address the role of our novel anti-GARP antibody in therapeutic efficacy against NSCLC and elucidate the mechanisms by which it regulates CD8+ T cell programming and effector function. It has the potential to overcome risks associated with systemic inhibition of TGFβ pathway and produce a novel agent for cancer immunothera...