Simultaneous measurement of multiple biomarkers such as cytokines, chemokines, growth factors, cell death, and cancer markers has gained increasing popularity in drug development. Testing multiple biomarkers in a single assay can not only reduce the amount of sample volume and hands-on time required but also allow assessment of biomarkers across many types of targets/pathways as a more comprehensive snapshot of the disease state and/or the study subject’s response to drug treatment, thereby accelerating the research process. However, developing a multiplex assay is a highly challenging endeavor, demanding smart decision-making and prioritization to balance many parameters to create a practical and useful tool for translational research.
There are two objectives of our presentation:
First, the development of two home-brew U-plex multiplex panels, which are to measure chemokines/chemokines associated with immune-activation, and to measure cancer biomarkers, respectively, will be presented. These two case studies will provide an overview of challenges and key considerations for a successful multiplexing development. Many factors, such as antibody pair specificity, multiplex-ability, assay quantitative ranges, and choice of sample dilution and diluents, and the experimental details and outcomes will be presented. As a result, two home-brew U-plex assays, one panel with 7 chemokines and another with 5 cancer markers were successfully developed and validated on the MSD (Meso Scale Discovery) platform using its U-plex technology. The chemokine panel includes CCL-2 (MCP-1), CCL-3 (MIP-1a), CCL-4 (MIP-1b), CCL-19 (MIP-3b), CXCL-9 (MIG), CXCL-10 (IP-10) and CXCL-11 (ITAC), whereas the cancer biomarker panel includes CA125, CA15.3, CEA, Osteopontin (OPN) and PD-1.
Second, in addition to developing and pre-study validating the assay, monitoring the assay performance in the long run becomes essential for ongoing clinical studies, especially when reagent lots changed during the study. Several approaches and practices will be shared in the presentation to address the long-term performance of the multiplex assay, which includes evaluating the performance of the recombinant analytes, wild-type control, and in-study sample re-analysis. By implementing these measures, we can minimize the influence of assay drifting or reagent variability and ensure that the observed changes in analytes truly reflect the physiological or disease status changes upon drug treatment.
These two assays have been successfully developed and validated. In the past 4 years, the assays were widely used for supporting many I-O and oncology programs and for testing more than 7000 clinical study patient samples for exploratory purposes. Home-brew U-plex assays were proved to be highly valuable tools for evaluating the mechanism of action and pharmacodynamic effect of investigational drugs, and for monitoring cancer diseases.
Learning Objectives:
Understand the benefits of multiplex and different multiplex platforms
Understand the challenges and key considerations for a successful "homebrew" multiplexing project
Understand some of the key factors and how to prioritize and balance them to develop a multiplex assay with an overall better performance of all analytes
Learn a systemic approach to monitor multiplex assay performance for ongoing clinical studies.
