(T0930-06-37) Highly Sensitive Analysis Using EVOSEP-LC/MS Assay for Targeted PD-L1 and PD1 Expression Level for Predicting Response to Immune Checkpoint Inhibitors

Purpose: The use of monoclonal therapeutic antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. PD-L1 and PD1 immunoassays have been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, PD-L1 and PD1 as biomarkers show limitations. Among them, there is no consensus about the antibodies used to determine PD-L1 and PD1 expression levels leading to uncertainty about the universal clinical value of these biomarkers across tumor types. In this work, we set to test a robust method to determine PD-L1 and PD-1 expression levels as compared to routinely used immunoassay at the analytical level and then use the Overall Response Rate (ORR) to assess clinical utility.

Methods: Tumor tissues samples from resected non-small cell lung cancer (NSCLC) patients further treated with adjuvant pembrolizumab therapy were assessed for PD-L1 and PD1 expression by Evosep-LC/MS and ELISA immunoassay with different antibodies clones. A subset of serially diluted cases was evaluated for Evosep-LC/MS assay performance across a range of PD-L1 and PD1 expression levels shown to correlate with the Objective Response Rate (ORR) of the patients: Complete Response (CR) and Progression Disease (PD).

Results: Assessment of PD-L1 and PD1 protein levels by Evosep-LC/MS demonstrated robust linearity across high and low expression range. Reproducible protein expression levels were obtained for Evosp-LC/MS and the different ELISA kits tested, and both technologies delivered broadly comparable titration values. Still, for a given tissue dilution, the actual expression level of PD-L1 and PD1 varied between the different ELISA kits. Targeting a given antibody clone biases the actual expression level evaluation, whereas such a bias is not observed from a label-free quantification assay such as the Evosep-LC/MS one. Evosep-LC/MS has the added advantages of being amenable to standardization and avoidance of interpretation bias for clinical use.

Conclusion: Equivalent sensitivity was established between EVOSEP LC-MS/MS and ELISA methods with slightly greater precision for the LC-MS/MS. This methodology provides a harmonized high throughput testing for PD-L1 and PD1 biomarkers for predicting clinical response.