Background: The limitations of current biomarkers for predicting outcome to anti-PD1 therapy, such as measuring tumor PDL1, are well-known. Evaluation of the PD1-PDL1 interaction as a potential biomarker has not been explored because of the lack of a suitable assay. We previously reported on a novel technology called the LIRECAP (Ligand-Receptor Complex-binding Aptamer) assay that quantifies the fractional occupancy of a receptor by its ligand (PMID: 31383650). LIRECAP assay is based on two RNA aptamers with differential binding to the unoccupied receptor vs. the ligand-receptor complex, where the ratio of aptamer binding reflects fractional occupancy. Here, we report the generation of RNA aptamers for use in a LIRECAP assay to quantify PD1-PDL1 interactions in tumor.

Methods: SELEX: RNA aptamers were enriched using a variation of SELEX that consisted of pre-clearing followed by a positive selection against human PD1, PDL or the PD1-PDL1 complex. Target-bound aptamers were extracted, amplified with SEL2 primers and transcribed. After eight rounds, enriched aptamers were sequenced and lead aptamers were identified. Assessing target binding: Dynabeads were coated with recombinant PD1 alone or PDL1 alone. Complex-coated beads were made by incubating PD1-bound beads with PDL1. Beads were formalin-fixed to mimic biospecimens and prevent PD1-PDL1 dissociation. Aptamers were added to beads, unbound aptamers washed off and bound aptamers extracted and quantified by SYBR green-based RT-qPCR using SEL2 primers. Binding of each aptamer was normalized to the binding of unselected aptamer library (Rd0).

Results: (1) A progressive enrichment of unique sequences was seen in all the three SELEX (PD1, PDL1, Complex) with maximum enrichment reached between rounds 4 and 5. (2) Binding of aptamers: Most of the lead candidates showed predominant binding to the targets against which they were enriched. (a) Three out of four lead aptamers from the PD1 SELEX showed significantly higher binding to unoccupied PD1 than to PD1-PDL1 complex (p<0.05). (b) Two out of five lead aptamers from the PDL1 SELEX showed significantly higher binding to unoccupied PDL1 compared to the complex (p<0.05). (c) Four out of five lead aptamers from the Complex SELEX showed significantly higher binding to PD1-PDL1 complex than to unoccupied PD1 (p<0.05). None of these aptamers showed cross-reactivity to unoccupied PDL1.

Conclusions and Significance: Novel RNA aptamers with differential specificity towards PD1, PDL1 or PD1-PDL1 complex have been successfully created for use in a LIRECAP assay that will be used to quantify the fractional occupancy of PD1 by PDL1 in tumor biospecimens. Studies assessing whether the resulting assay can predict response to anti-PD1 therapy are expected to begin shortly.

Citation Format: Suresh Veeramani, Travis Fischer, William H. Thiel, George J. Weiner. Generation of RNA aptamers for measuring fractional occupancy of PD1 by PDL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2804.