Abstract 4586: Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models

T regulatory (Treg) cells play an important role in maintaining immunological tolerance to self-antigens, thus limiting immune responses to tumor antigens. Therefore, depleting or suppressing Tregs is one strategy by which anti-tumor immunity can be restored. The immunotoxin ONTAK® is an IL-2-diphteria toxin fusion protein that has been shown to diminish Tregs in patients and animal models of cancer in peripheral blood using a systemic intravenous (i.v.) administration route. E7777 is a new version of ONTAK®. In this study we tested the hypothesis that locally-diminished Tregs by intratumoral (i.t.) administration of E7777 generate effective anti-tumor immune response at both local and systemic levels. First, we showed superior anti-tumor activity and safety of E7777 i.t. over E7777 i.v., where i.t. administration resulted in complete tumor regressions in both moderately immunogenic CT26 and non-immunogenic B16F10 tumors with minimal animal body weight loss. In contrast, only tumor growth delay was observed for E7777 i.v. with dose-limiting animal body weight reduction in the same models. Immune phenotyping showed a 4 fold reduction of intratumoral Tregs in treated CT-26 tumors without significant change of Tregs in the spleens of treated animals, confirming a local Treg-depleting effect of E7777 i.t. In contrast, intratumoral CD8+ T cells were not reduced. Second, E7777 i.t. enhanced overall anti-tumor immune response, manifested by significantly increased numbers of CD45+ hematopoietic cells, Granzyme B+ CD8+ cytotoxic T cells, and ratios of cytotoxic T cells/Tregs in the treated tumors. Importantly, E7777 i.t. also resulted in distant effects in the spleen characterized by increased ratio of T lymphocytes to myeloid cells and increased frequencies of both effector memory CD8+ T cells (CD8+CD62L-CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) indicative of systemic immune activation. Consistent with the generation of immunological memory, 60% of the tumor-free animals treated with E7777 i.t. rejected completely and 40% displayed delayed tumor growth of B16F10 cell challenge while all naïve control animals grew tumors. Taken together, our results demonstrate that intratumoral Treg depletion by local administration of E7777 leads to an effective local and memory anti-tumor response in preclinical models and support further evaluation of local E7777 delivery as a cancer immunotherapy. Citation Format: Diana I. Albu, Christy Ingersoll, Kuan-Chun Hua