(M1530-09-52) Validation of a High-Sensitivity Inflammatory Cytokine Panel to Improve Clinical Biomarker Data Utility

Purpose: Biomarkers have become an increasingly important driver in the development of pharmaceutical therapeutics; informing on biological activity, efficacy, and safety. Biomarker data can also be used as a tool for patient enrollment and stratification in clinical trials to better target the responsive population. Depending on the context of use, it may be necessary to measure these biomarkers in combination, at low concentrations to evaluate the therapeutic benefit. These data have been challenging to generate, as ELISA and other conventional platforms require large volumes of matrices, typically measure single analytes, may perform with low accuracy and precision, or unable to resolve low concentrations of analytes to sufficiently distinguish relevant clinical cutoffs (Monastero, 2017). To address these challenges, a highly sensitive multiplex electrochemiluminescence (ECL) method for measuring a panel of immune-derived biomarkers: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17α and TNF-α in human plasma and serum samples was recently validated using the Mesoscale Discovery (MSD) S-Plex Proinflammatory Panel 1 platform. These inflammation and immune system regulating biomarkers have been shown to have high prognostic utility across various therapeutic areas including but not limited to: HIV (Silva, 2020), major depression (Hocaoglu, 2012), and oncological indications (Floros, 2015, Kursunel, 2017). The validated assay was shown to simultaneously measure these analytes using low sample volume, precisely and accurately into fg/mL ranges. The assay was validated using the principle of current regulatory guidance criteria for ligand binding methodologies (FDA, 2018, Piccoli, 2019).

Methods: Assay kits were procured from Mesoscale Discovery MSD (Gaithersburg, MD). Normal human serum and plasma were procured from BioIVT (Westbury, NY). Calibration curves were prepared according to manufacturer’s instructions and assayed in duplicate wells over 8 individual runs. Validation samples were prepared in pooled serum and plasma, with spiked calibrator to target concentrations spanning the standards ranges. The samples were run over 7 individual runs by 4 analysts. For selectivity assessments, ten individual human and serum samples were spiked with calibrators. To assess linearity, four serum and four plasma individuals were spiked with calibrator and diluted to 5 concentrations spanning the quantitative ranges. Prozone effect was assessed by preparing analytes at 10X the calibrator ULOQ, and diluted to within the quantitative ranges. Stability was assessed by spiking calibrators into each matrix at 2 concentrations, and subjecting 5 freeze-thaw cycles, storage at room temperature (RT) or refrigerated (4C) overnight (approximately 19 hours).

Results: Calibration ranges were based on highest and lowest concentrations of calibrators meeting precision and accuracy acceptance criteria, and were determined to be (fg/mL): IFN-γ: 31,700 - 7.74, IL-1β: 168,000 - 41.0, IL-2: 80,200 - 19.6, IL-4: 54,200 - 13.2 IL-6: 53,900 - 13.2 IL-10: 174,000 - 42.5 IL-12p70: 486,000 - 118.7 IL-17α :259,000 - 63.2 TNF-α: 52,600 - 12.8. Quantitative ranges were based on highest and lowest concentrations of validation samples meeting precision and accuracy acceptance criteria, and were determined in human serum as (fg/mL): IFN-γ: 24,429 - 31.3, IL-1β: 108,291 - 77.0, IL-2: 80,474 - 27.9, IL-4: 31,353 - 17.6 IL-6: 33,488  104.3 IL-10: 63,206 - 47.3 IL-12p70: 366,551 - 131.2 IL-17α :169,901 - 87.2 TNF-α: 37,470 - 18.1 and in human plasma as: IFN-γ: 24,517 - 13.8, IL-1β: 141,009 - 69.5, IL-2: 96,724 - 31.5, IL-4: 43,242 - 26.3 IL-6: 42,981 - 80.9 IL-10: 127,152 - 114.7 IL-12p70: 464,365 - 212.3 IL-17α: 198,218 - 134.3 TNF-α: 47,108 - 32.0. The assay was determined to be selective for all analytes, with precision assessments being met between all individuals. Linearity was found to be acceptable for all analytes and dilutions, with precision assessments being met between in all subjects and both matrices. No prozone effect was observed in any of the analytes, with all dilution series results showing a linear relationship. Stability assessments in human serum were acceptable overnight at room temperature except the high samples for IL-1β, and IL-17α, and the low samples for IL-2, and IL-10. At 4C, serum samples were acceptable except for high IL-1β. Serum samples with 5 freeze thaws were acceptable except for high IFN-γ, IL-2, IL-10, and IL-12p70 and low IL-10. Plasma samples were acceptable overnight at room temperature high IFN-γ, IL-1β, and IL-17α. At 4C, all plasma samples were acceptable except high and low IL-1β. The high and low plasma samples with 5 freeze thaws did not pass for IL-1β, IL-10, IL-17α, and TNF-α, or the high sample for IFN-γ.

Conclusion: With the use of this validated biomarker assay, we can more accurately and efficiently resolve biomarker data involving these inflammatory pathways. These data will help to better define ranges between normal and disease states with higher confidence. Additionally, the measurements were able to be validated using low volume of matrix, resulting in a decreased burden upon patient sampling. Ultimately, these improved biomarker data will help to transition therapeutics from clinical trials to approval, where they can benefit patients in need.

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Validation Executive Summary of Results