Abstract 3267: Cancer immunotherapy by use of FANA ASO therapy to silence Foxp3, impair Treg function and promote anti-tumor immunity

There is an inverse correlation between tumor and draining lymph node infiltration by Foxp3+CD4+CD25+ Treg cells and clinical outcomes in carcinomas of the breast, liver, stomach, ovary, pancreas, and lung, as well as melanomas and glioblastomas. Tregs suppress tumor responses by host CD8 and CD4 T cells, B cells, NK cells, macrophages and dendritic cells. There are currently no effective ways to target Tregs, since toxins (e.g. IL-2-diptheria toxin) and cyclophosphamide have only transient effects with rebound and may also affect conventional T cells, use of CD25 monoclonal antibodies have confounding effects given IL-2 receptor (CD25) expression by activated T cells, and given the phenomenon of “peripheral” conversion (iTreg development) at the tumor site that can allow Tregs to evade therapy. These problems led us explore use of FANA antisense oligonucleotides (ASO) to suppress expression of Foxp3 mRNA. FANA ASO are highly specific and are taken up by cells without use of any delivery agents, conjugates or formulations, making them attractive compounds for cancer drug development. Various Foxp3 sequence-specific or scrambled FANAs were designed, and fluorescently-labeled FANA tested by addition to Foxp3+ Tregs in vitro; these studies showed successful uptake and both cytoplasmic and nuclear localization. FANAs were further screened by assessing effects on Treg suppressive function, and those resulting in 40-50% reductions were assessed for effects on expression of Foxp3 mRNA (qPCR) and protein (Western blots). We next tested the in vivo efficacy of Foxp3 FANA by intravenous or intraperitoneal injection, either daily or twice/week, of Foxp3 FANA, followed by isolation of Tregs from lymphoid tissues. Selected fluorescently-labeled FANA showed Treg uptake, and unlabeled FANA led to >75% reduction in Foxp3 protein expression (flow cytometry, Western blots), and marked reductions in Treg suppressive function. Lastly, we compared the effects of scrambled vs. specific Foxp3 FANA ASO on TC1 lung tumor growth in syngeneic C57BL/6 mice. Compared with mice receiving scrambled FANA ASO, mice receiving specific Foxp3 FANA ASO showed impaired tumor growth, with 50% showing complete tumor elimination (p Citation Format: Wayne W. Hancock, Liqing Wang, Veenu Aishwarya. Cancer immunotherapy by use of FANA ASO therapy to silence Foxp3, impair Treg function and promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3267.