Abstract 3225: Development of a proximity-based immunoassay to measure the PD1:PD-L1 complex in fixed samples
Authors: Wallweber Gerald , Ravanera Roy , Chenna Ahmed , Stathas David , Huang Weidong , Winslow John , Petropoulos Christos 
Published: July 2016

Abstract
Introduction: The binding of tumor cells expressing PD-L1 to PD1 receptors on activated T-cells inhibits T-cell activation, thereby evading an immune response against tumor progression. Currently, therapeutic monoclonal antibodies directed against either PD1 or PD-L1 block this interaction and restore T-cell function, leading to tumor lysis. PD-L1 immunohistochemistry (IHC) is the most common biomarker assay for these therapies, but disagreement exists on the antibodies used for IHC, which cell types to score, and the criteria for positivity. Quantitative immunofluorescence identified a significant correlation between the proximity of PD1 and PD-L1 protein expression and response to anti-PD1 therapy (Tumeh et al., Nature. 2014; 515:568-71). We have developed a direct quantitative measurement of the PD1:PD-L1 complex for the stratification of patients for anti-PD1 or anti-PD-L1 therapy. The VeraTag PD1:PD-L1 complex immunoassay utilizes two antibody pairs for the proximity-dependent release of a fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately quantify the amount of complex in fixed samples.
Methods: Anti-PD-L1 rabbit mAb E1L3N was selected from our previously established PD-L1 VeraTag immunoassay. Mouse anti-PD1 mAbs were screened against FFPE human tonsil and PBMCs for development of a PD1 VeraTag assay. The VeraTag assay for PD1:PD-L1 complex combined the anti-PD-L1 rabbit mAb E1L3N and the anti-PD1 mouse mAb NAT105 together with a goat anti-rabbit secondary Ab conjugated to the VeraTag reporter and a goat anti-mouse secondary Ab conjugated to biotin. Fixed cell preparations used in the VeraTag PD1:PDL1 complex assay were prepared by co-culturing MB453, MB231 or RKO cancer cell lines expressing varying amounts of PD-L1 with Jurkat T cells. After 48-hours, non-adherent cells were removed by washing and remaining cells were fixed overnight in formalin.
Results: Following phytohemagglutinin (PHA) stimulation of human PMBCs and Jurkat cells, VeraTag measurements of PD1 protein expression increased 8- to 10-fold, whereas PD-L1 protein expression varied &lt;2-fold under the same conditions. VeraTag measurements for the PD1:PD-L1 complex increased approximately 4-fold with the addition of PHA to co-cultures of PD-L1+ cancer cell line and Jurkat T cells. The level of PD1:PD-L1 complex varied proportionally with the number of Jurkat T cells added to the co-culture. We are currently utilizing VeraTag assays to quantify the amounts of PD1, PD-L1 protein and the PD1:PD-L1 complex with the VeraTag assays across a panel of formalin fixed samples of breast, lung and squamous cell carcinoma of the head and neck (SCCHN) cancer samples.
Conclusions: We have developed sensitive and quantitative assays for PD1, PD-L1 and the PD1:PD-L1 complex, which may provide a more direct measurement of PD1/PD-L1 pathway interaction.
Citation Format: Gerald Wallweber, Roy Ravanera, Ahmed Chenna, David Stathas, Weidong Huang, John Winslow, Christos Petropoulos. Development of a proximity-based immunoassay to measure the PD1:PD-L1 complex in fixed samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3225.