Abstract B057: Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown
Authors: Zhang Ying , Lieberman Judy 
Published: February 2019

Abstract
Triple-negative (TNBC) and HER2+ breast cancers (BCs) are especially aggressive tumors, which are prone to relapse and metastasize post chemo- or targeted therapy. Immunotherapy provides a promising, but unproven, alternative approach for treating poor-prognosis BCs. BCs have relatively low nonsynonymous mutation rates, suggesting that many BCs will respond poorly to immunotherapy. Novel strategies to increase BC cell immunogenicity and improve tumor-antigen specific T-cell responses are needed to enhance the efficacy of BC immune therapy. We aim to enhance the immunogenicity of breast tumor by taking advantage of the unique strength of EpCAM-aptamer conjugated small interfering RNAs (AsiCs), which can knock down gene expression selectively in EpCAM+ BC cells, to make aggressive BCs visible to T-cells and improve T-cell tumor recruitment and function. EpCAM is a tumor-specific antigen expressed at several logs higher levels on all epithelial cancers and cancer stem cells relative to normal epithelia. The EpCAM aptamer our lab developed binds with high affinity to both mouse and human EpCAM. Using this platform, we investigated the use of EpCAM-AsiCs for BC immune modulation by targeting genes controlling different functional processes: 1) knocking down the nonsense-mediated mRNA decay (NMD) gene UPF2, to elicit tumor neoantigen expression to improve BC tumor recognition by the immune system; 2) knocking down CD47 to promote phagocytosis of cancer cells and enhance cross-presentation of tumor antigens; 3) knocking down DNA repair enzyme PARP1 to produce more DNA damage and consequent genetic mutations, thereby introducing tumor-specific neoantigens to the immune system; 4) knocking down PLK1 which is essential for cell mitosis, and MCL1, which is a critical survival factor in TNBC, to directly kill tumor cells. The increased tumor cell death induced by PLK1 and MCL1 knockdown could potentially promote tumor antigen cross-presentation to CD8+ T-cells. Our data indicate that each of these AsiCs potently silences gene expression within mouse or human BC cell lines in vitro, and efficiently reduce target gene expression in EpCAM+ tumor cells in vivo. When treating mice bearing 4T1E (4T1 cell line with high EpCAM expression) tumors, EpCAM-AsiCs targeting UPF2, CD47, PARP1, PLK1 and MCL1 each significantly suppressed 4T1E tumor growth, increased CD8+ T-cell tumor infiltration, CD8+ T-cell/regulatory T-cells (Treg) ratio, the effector functions and degranulation capacity of CD8+ TILs compared to those of the control group. The capacity of these CD8+ TILs to directly kill tumor cells was also improved. Tumor-associated macrophages (TAMs) from CD47-AsiC treated tumors showed improved phagocytic capacity as determined by in vitro and in vivo phagocytosis assays. CD47-AsiC also increased the ratio of M1 (tumor suppressive) to M2 (tumor promoting) TAMs. Dendritic cells (DCs) from CD47-AsiC treated tumors show enhanced CD40 and CD86 expression, suggesting an improved immunostimulatory capacity. Using antibody depletion assay, we further show that CD4+ T-cells, CD8+ T-cells and macrophages all contribute to the tumor inhibition capacity of CD47-AsiC; and TAMs/DC play an important role in stimulating the functions of CD8+ TILs. In addition, we show that CD47-AsiC outperforms the anti-CD47 antibody in suppressing 4T1E tumor progression. Finally, we show that UPF2, CD47, PARP1, PLK1 and MCL1 AsiCs work in synergy to further delay tumor growth, and even lead to tumor regression, compared to single AsiC treatment. The combination therapy significantly increased the amount of CD8+ TILs, reduced the Tregs and myeloid-derived suppressor cells (MDSCs) in the tumor, and improved the functions of both CD4+ and CD8+ TILs. Our data suggest that these novel EpCAM-AsiCs, with their small size and high selectivity, have great potential to improve therapeutic efficacy and reduce toxicity for BC patients compared to current checkpoint inhibitors.
Citation Format: Ying Zhang, Judy Lieberman. Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B057.