{
  "abstract": "Background Existing biomarkers used to predict clinical response to anti-PD1 therapy are suboptimal. Here, we report on a novel assay, known as the PD1 Ligand-Receptor Complex-binding Aptamer (LIRECAP) assay designed to quantify the fractional occupancy (FO) of PD1 by PDL1 in clinical formalin fixed paraffin embedded (FFPE) biospecimens.Methods PD1 LIRECAP assay: The PD1 LIRECAP assay was developed by identifying a pair of RNA aptamers with one binding preferentially to unoccupied PD1 (P aptamer) and the other to the PD1-PDL1 complex (C aptamer). For the PD1 LIRECAP assay, an equimolar mix P and C aptamers are added to a biospecimen and bound aptamers were quantified simultaneously using a two color TaqMan-based RT-qPCR with a single set of PCR primers and two unique TaqMan probes specific for each aptamer. The ratio of bound aptamers (C:P ratio) reflects PD1 FO in that biospecimen (figure 1). Co-culture system: The PD1+Luciferase+ Jurkat – PDL1+ Raji cell co-culture system (Invivogen) was used to compare the results of the PD1 LIRECAP to PD1 signaling. Jurkat cells were cocultured with varying numbers of Raji cells expressing PDL1 to create a range of PD1 FO (0 to 100%). PD1 signaling, indicated by the inhibition of luciferase activity in Jurkat cells, was determined by bioluminescence in the conditioned media. Cells from the same samples were formalin fixed and used for the PD1 LIRECAP assay. Sarcoma biospecimens: Six FFPE sarcoma biospecimens were grouped as PDL1lowT cellhigh (n=3) and PDL1highT cellhigh (n=3). Sections were cut from each biospecimen for the PD1 LIRECAP assay and a proximity ligation assay (PLA).Results Co-culture system: PD1 signaling, as measured by inhibition of luciferase activity, and C:P ratio, as measured by the PD1 LIRECAP correlated with increasing PD1 FO (R 2=0.961 and 0.896, respectively). C:P ratio and PD1 signaling also correlated with each other (R2=0.77; p=0.022).Sarcoma biospecimens. The PD1 LIRECAP assay on clinical biospecimens was technically highly reproducible. PDL1highT cellhigh tumors showed a significantly higher C:P ratio, indicating higher PD1 FO, when compared to PDL1lowT cellhigh tumors (p=0.01). PD1 FO also correlated with the proximity of PD1 and PDL1 as evaluated by PLA.Conclusions PD1 LIRECAP assay represents a novel approach to quantifying PD1 FO in FFPE clinical biospecimens. Studies assessing the value of PD1 FO as a predictive biomarker as determined by the PD1 LIRECAP assay are ongoing.Ethics Approval The study utilized de-identified tumor biospecimens, as approved by the University of Iowa.Abstract 112 Figure 1Schema of the PD1 LIRECAP assay",
  "authors": [
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "Suresh Veeramani"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "Chaobo Yin"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "Kristen Coleman"
    },
    {
      "affiliations": [
        "University of Iowa, Iowa City, IA, USA"
      ],
      "name": "George J Weiner"
    }
  ],
  "title": "112 Quantifying the fractional occupancy of PD1 by PDL1 in clinical biospecimens using a novel RNA aptamer-based assay",
  "uid": "73578bd3-5216-53f5-9dbf-ba5deb8d7a35"
}