Abstract LB163: Novel multiplex immunoassay for simultaneous profiling of human and mouse immunomodulatory proteins in humanized models

Abstract Multiplex immunoassay kits are essential for profiling a wide array of immunomodulatory proteins, including cytokines, chemokines, and growth factors in humans and animal models. Humanized mice are genetically modified to express human genes or engraft human cells, allowing researchers to study human immune responses, vaccine efficacy, and immune-related diseases. However, the high cross-species homology of many immune-related proteins complicates the distinction between human and mouse homologues within the same sample. Therefore, reliable tools that can differentiate these homologues are crucial for researchers using humanized mouse models. We describe the development of a novel multiplex immunoassay featuring both mouse-specific and human-specific antibody pairs against cytokines, chemokines, and growth factors. Specificity was confirmed by testing individual mouse and human recombinant proteins. This kit was optimized for mouse serum and plasma samples, detected by mouse-analyte capture beads, while human-analyte capture beads showed no reaction. To evaluate the platform's ability to measure human cytokines, we spiked human PBMC supernatant into mouse serum and plasma samples. The exogenous human analytes from the PBMC supernatant were specifically detected on the human beads in the spiked mouse serum. We further validated this platform by analyzing serum collected from immunodeficient NSG mice bearing human glioma xenografts treated with tumor-targeting or non-targeting human CAR T cells. Peripheral levels of human interferon gamma were lower in cohorts treated with locoregional administration of CAR T cells compared to cohorts receiving systemic administration of CAR T cells, highlighting the real-world applicability of this novel multiplex immunoassay kit. In summary, this study demonstrates the feasibility and utility of measuring mouse and human proteins simultaneously via multiplex immunoassay, providing a powerful tool for advancing research in humanized mouse models. Citation Format: Anthony J. Saporita, Jonathan F. Khan, Tina Raeber, Brice Martin, Parwiz Abrahimi, Brooke Gilliam, Shane Curran, Rick Wiese, Mark M. Souweidane, Jedd D. Wolchok, Taha Merghoub. Novel multiplex immunoassay for simultaneous profiling of human and mouse immunomodulatory proteins in humanized models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB163.