Background: There is a need for better prognostic biomarkers for PD1-based therapy. Quantifying the fractional occupancy (FO) of PD1 by PDL1, and PDL1 by PD1, in tumor can be a potential biomarker. We previously reported on an RNA aptamer-based assay platform called the Ligand-Receptor Complex Aptamer (LIRECAP) assay that quantifies FO of a receptor by its ligand. Here, we describe a 3-color LIRECAP assay designed to quantify PD1 occupancy by PDL1 and PDL1 occupancy by PD1 in formalin fixed paraffin embedded (FFPE) tissue.

Methods: (A) Selection of aptamers: RNA aptamers with preferential binding to PD1 (“P” aptamers), PDL1 (“L” aptamers) or PD1-PDL1 complex (“C” aptamers) were selected from single starting library using a variation of the SELEX, we reported previously (PMID: 31383650). Selected aptamers were tested for binding to formalin fixed proteins that stabilized the complex and assured that selected aptamers bound to the targets in FFPE biospecimens. Lead aptamers, namely P17, L42 and C113, were selected for further study based on their preferential binding to formalin fixed PD1, PDL1 and complex respectively. Three TaqMan-based calorimetric probes were designed targeting the unique central regions of P17, L42 and C113. (B) 3-color LIRECAP assay on FFPE tissue: Cell culture samples containing PD1, PDL1 or PD1-PDL1 complex were created by mixing equal numbers of (1) PD1+ Jurkat with PDL1- Raji cells, (2) PD1- Jurkat with PDL1+ Raji cells or (3) PD1+ Jurkat with PDL1+ Raji cells, respectively. Cell blocks were produced using the collodion bag technique, fixed in formalin and embedded in paraffin using standard clinical techniques. Scrolls of 10-um were cut, deparaffinized, rehydrated, washed and non-specific binding blocked. An equimolar mix of P17, L42 and C113 aptamers was added, bound aptamers were extracted and their relative concentrations were quantified simultaneously by 3-color TaqMan-based RT-qPCR using a single set of primers. The proportion of each aptamer was calculated as the percentage of each aptamer (P or L or C) relative to the amount of total bound aptamers (P+L+C).

Results: Each aptamer bound preferentially (underlined) to its designated target as determined by 3-color assay (mean of 2 independent sets).

Unoccupied PD1 only: P17=44%; L42=34%; C18=22%

Unoccupied PDL1 only: P17=24%; L42=48%; C18=28%

PD1-PDL1 complex: P17=19%; L42=17%; C18=64%

Although preferential binding of aptamers was not absolute, simultaneous evaluation of three aptamers in a single assay provided an internal control that can allow for correction for this cross-reactivity.

Conclusions and significance: The 3-color LIRECAP assay is capable of quantifying FO of PD1 by PDL1 and PDL1 by PD1 in FFPE samples. Ongoing studies are exploring use of this assay to quantify FO in clinical biospecimens. It will then be used to determine whether PD1 FO might serve as a novel predictive biomarker for anti-PD1 therapy.

Citation Format: Suresh Veeramani, Nanmeng Yu, Xian Jin Xie, Kristen Coleman, George J. Weiner. A novel RNA aptamer-based assay for quantifying fractional occupancy of PD1 by PDL1 in formalin fixed biospecimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3313.