Background: 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 allowing tumors to escape immune surveillance. Recently, several antibodies that inhibit the signaling from PD-1/PD-L1 axis were developed that showed enhanced anti-cancer immune reponse leading to their FDA approval for therapeutic use. Aptamers are synthetic small molecule ligands composed of short, single-stranded RNA or DNA oligonucleotides with specific 3D conformation. Aptamers mimic antibodies to recognize and bind to their targets with high affinity. 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. FDA has approved several aptamers for therapeutic use that include Macugen, a RNA aptamer that inhibits VEGF signaling to treat age-related macular degeneration, Vitravene to treat cytomegalovirus (CMV) retinitis and Kynamro to treat homozygous familial hypercholesterolemia. There are several other potentially promising aptamers in ongoing preclinical and clinical tests.

Methods: 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.

Results: 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 pM 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. In vitro cytotoxicity T- cell assays demonstrated the ability of PD-1 aptamer to potently enhance T-cell response and cytokine production in MLR. Our findings from pharmacokinetics in vivo studies demostrated that PEG-conjugation of PD-1 aptamer has improved the stability of DNA in mouse plasma. A PEGylated form of PD-1 DNA aptamer has demonstrated the ability to block PD-1:PD-L1 interaction and significantly reduced the growth of HEL92.1.7 leukemic tumor cells in vivo in humanized NSG mice with a potency equivalent to an antagonistic anti-PD-1 therapeutic agents. Tumor growth inhibition in humanized NSG mice treated with PD-1 DNA aptamer was 22.59% and comparable to PD-1 antibody over the control group.

Conclusions: Overall, our findings demonstrate a comprehensive preclinical characterization of PD-1 aptamer for which antitumor activity was demonstrated in mouse studies.


Iyer: Genentech: Research Funding; Takeda: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.