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Aptamers are oligonucleotide or peptide molecules that bind to a specific target with high affinity and specificity. A diverse range of modifications have been reported for either enhancing aptamers’ nuclease resistance or expanding their chemical functionalities. We created a combinatorial DNA aptamer library that had been modified with thio substitution of the phosphate backbone (thioaptamer, TA) at selected 5’dA positions for cell and tissue based selections. Based on morphological assessment of the tissue, we used an image-directed laser microdissection (LMD) technique to dissect Regions of Interest (ROIs) bound with thioaptamers, and further identified the targeted proteins. Using a Next Generation Sequencing (NGS) approach and alignments by our recently developed Aptaligner program, we are able to select tissue specific TAs in a more representative way from the TA libraries. We have successfully identified TAs that bind specifically to tumor endothelial cells and characterized the lead candidate TA, Endo28, as an Annexin A2-specific TA that allows for selective delivery to the tumor vasculature. Based on the primary sequences of several of the previously selected TAs and observed variations, we developed an X-aptamer (XA) library by conjugating drug-like ligands (X) to the DNA bases in a random fashion. We were able to select an XA with high affinity to CD44 hyaluronic acid binding domain (HABD) from a large combinatorial TA library modified with a drug (N-acetyl-2,3-dehydro-2- deoxyneuraminic acid, ADDA). The selected CD44 X-aptamers (amine form and ADDA form) show enhanced binding affinities with ADDA to CD44 over-expressed on human ovarian cancer IGROV cells. Using a systems biology approach that combines the use of 15 mm bead-based XA libraries with flow cytometry sorting and mass spectrometry, we collected beads binding to proteins labeled with different fluorescence dyes and identified their associated XA sequences by PCR and NGS. The differentially expressed proteins can be identified by an XA beads pulldown experiment to discover the potential biomarkers for ultimately TA or XA targeted nanoparticle delivery of drugs to tumors.

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

Anti-angiogenesis targeted therapy using RNA interference is a powerful tool to inhibit tumor growth and metastasis Here, we developed chitosan/thioaptamer nanoparticles that are capable of cell-type specific binding and delivery of siRNA into tumor-associated endothelial cells Thioaptamer (TA) was created by partial substitution of oxygen with sulfur on the aptamer phosphate backbone at the 5′dA position to enhance binding affinity and stability A combinatorial DNA aptamer library was established for cell-based selection method known as SELEX (systematic evolution of ligands by exponential enrichment) Endothelial cells isolated from both human ovarian tumors and normal human ovarian tissues were used to screen for selective thioaptamers After 10 cycles of cell-SELEX, two candidates, Endo28 and Endo31 showed highly specific binding to both human microvascular endothelial cells (HMVEC) and vasculature of human ovarian cancer tissue Endo28 decorated chitosan nanoparticles (CH/TA-siRNA) were ∼200 nm, positively charged (22mV), and spherical in shape The incorporation of thioaptamer Endo28 and siRNA to chitosan was confirmed using 1HNMR and PCR CH/TA-siRNA nanoparticles were intact and stable in the presence of serum at 37° C Intravenous administration of CH/TA nanoparticles into mice with HeyA8 ovarian tumors resulted in successful binding of aptamer to endothelial cells as well as delivery of siRNA into tumors TEM7 is a tumor biomarker found on the surface of endothelial cells TEM7 silencing using siRNA reduced endothelial tube formation and migration In orthotopic mouse models of ovarian cancer, murine TEM7 siRNA loaded nanoparticles showed substantial reduction in tumor growth as well as reduced angiogenesis These data have implications for new anti-angiogenesis approaches Citation Format: Dahai Jiang, Lingegowda S. Mangala, Hongyu Wang, Sherry Wu, Lokesh G. Rao, Cristian Rodriguez-Aguayo, Sunila Pradeep, David E. Volk, Gabriel Lopez-Berestein, Anil K. Sood. Tumor vasculature targeting using cell-specific thioaptamer decorated chitosan nanoparticle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2014-4468