Abstract 3243: Development of specific DNA aptamers against programmed cell death-1 (Anti-PD1-Apt) for diagnosis and treatment of cancers

Cancer cells have an extraordinary ability to escape immune response by modulating proteins that regulate immune checkpoints and their cognate ligands. Programmed cell death protein (PD-1), a cell surface receptor expressed on T cells is one such immune checkpoint receptor when bound to its ligands- PDL1 or PDL2 transmits an inhibitory signal. Such modulation often leads to inhibition of T-cell activation and subsequent escape of tumors from immune surveillance. Recently, several FDA approved therapeutic antibodies have been successfully developed that target PD-1/PD-L1 axis and allow the immune system to enhance its anti-cancer effects. Aptamers are synthetic small molecule ligands composed of short, single-stranded oligonucleotides ranging from 30 to 60 bases in length. Based on their highly specific 3-dimensional conformation, aptamers, analogous to the antibodies can recognize and bind to their targets with high affinity. Also, the nucleic acid component has several advantages over the protein counterparts- such as ease of production under less stringent conditions, long shelf life and low cost. Here, we report the development of several PD-1 specific aptamers by systematic evolution of ligands by exponential enrichment (SELEX) technology against endogenous immunoprecipitated PD-1 protein using DNA library with a complexity of 1014. Following several rounds of SELEX, the selected aptamers sequenced by high throughput Next-Generation Sequencing (NGS) were found to have highly conserved regions. Six PD-1 specific aptamers (Anti-PD1-Apt) were then assessed for target validation using leukemic cell lysates (cell lines and primary patient samples) and were found to bind to the PD-1 in its native state. The selected Anti-PD1-Apt were able to specifically pull down the PD-1 protein from the lysates mimicking PD-1 antibody. The specific interaction of the Anti-PD1-Apt was also demonstrated by flow cytometry and fluorescent microscopy. As expected, Anti-PD1-Apt was able to bind to PD-1 with Kd of ~ 500 picomolar affinity as assessed by Bio-Layer Interferometry. Furthermore, we also characterized and confirmed Anti-PD1-Apt biological activity using an PD-1/PD-L1 cell-based assay using PD-1/NFAT reporter-Jurkat cells. We have observed several fold induction of NFAT luciferase reporter activity (Relative Luciferase Units) in PD-1/NFAT reporter-Jurkat cells co-cultured with HEK293 cells overexpressing PD-L1 and TCR activator in the presence of Anti-PD1-Apt compared to control. Our preliminary data also demonstrate robust Anti-PD1 blockade in Mixed Lymphocyte Reaction (MLR) along with induction of Th1 cytokines Interferon-gamma and IL-2 from different donor sets of PBMCs. We will present additional - in vivo antitumor response data at the upcoming AACR Annual Meeting at Washington DC, 2017. Citation Format: Santhana Gowri Thangavelu Devaraj, Lokesh Ganesh Lakshmana Rao, Youli Zu, Jenny C. Chang, Swaminathan P. Iyer. Development of specific DNA aptamers against programmed cell death-1 (Anti-PD1-Apt) for diagnosis and treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2017-3243