Abstract A112: A first in class immunotherapy that selectively activates the NFAT pathway in CD8+ T cells

Abstract Introduction: Despite the theoretic potential of immunostimulatory therapy in the treatment of cancer, the disruption of negative checkpoints in the tumor microenvironment remains the dominant clinical strategy. The failure of immunostimulation, utilizing agonistic cytokines or activating antibodies, can be attributed to off target side effects, the failure to preferentially activate cytotoxic lymphocytes (CTLs) over regulatory T cells (Tregs), and the development of T cell exhaustion resulting from Jak/STAT/PI3K signaling in T cells. Methods: To address these issues we designed a first in class immunostimulatory fusion protein (RB101) that utilizes NKG2D-targeting (without activation of the NKG2D signaling) to exclusively activate CTLs (CD8+ T, NK, γδ T cells). Previous approaches to target CD8+ T cells relied upon activation of the Jak/STAT/PI3K signal transduction pathway. In contrast, RB101 treatment results in selective activation of the nuclear factor of activated T cell (NFAT) signaling pathway. NFAT activation results in increased mitochondrial biogenesis, increased T cell receptor recognition of tumor antigens, accelerated proliferation, and augmented generation of memory T cells when compared to canonical cytokines and alternative methods of immunostimulation. Results: We observed that the unique combination of NFAT signaling and NKG2D-targeting led to CTL priming and expansion without systematic toxicity in a non-human primate (NHP) model. Subcutaneous RB101 administration to healthy cynomolgus macaques at or below the No Observed Adverse Event Level (NOAEL) increases the number of interferon gamma and granzyme B positive CD8+ T and NK cells 10-100-fold over vehicle treated animals. In addition, such administration resulted in >6-fold increase in CD8+ T/Treg and NK/Treg ratios, two leading indicators of clinical efficacy. Similar results were evident in human PBMC cultures in vitro. Subcutaneous RB101 administration results in partial and/or complete responses in mice bearing multiple syngeneic murine tumors. RB101 treatment also drives improved tumor control when combined with checkpoint inhibitors or radiation. Conclusion: The powerful immune response generated by signaling through the NFAT pathway has long been evident in organ allograft rejection. Clinical organ transplantation only became possible with clinical therapies targeting this signaling pathway (such as with tacrolimus). To our knowledge, RB101 is the first therapeutic designed to stimulate the NFAT pathway of T cell activation. The precise targeting of this stimulus to CTLs allows for the safe, subcutaneous administration of RB101 and results in a compelling preclinical profile of safety and efficacy in human cells and across multiple animal models. Citation Format: Eric Lazear, Dan Watkins, Alexander Krupnick, John Westwick. A first in class immunotherapy that selectively activates the NFAT pathway in CD8+ T cells [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A112.