Abstract 3243: Development of specific DNA aptamers against programmed cell death-1 (Anti-PD1-Apt) for diagnosis and treatment of cancers
Authors: Santhana Gowri Thangavelu Devaraj, Lokesh Ganesh Lakshmana Rao, Youli Zu, Jenny C. Chang, Swaminathan P. Iyer
Journal: Cancer Research (2017)

Abstract 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