Abstract LB165: Evaluating U5 snRNP200-targeting CAR-T therapy for acute myeloid leukemia: A novel multiplex immunoassay for analyte discrimination in mouse models

Abstract Multiplex immunoassay kits are essential for profiling immunomodulatory proteins, including cytokines, chemokines, and growth factors in humans and animal models. Humanized mice, engineered to express human genes or incorporate human cells, allow for the study of human immune responses and vaccine efficacy. The ability to distinguish human and mouse homologues in the same sample is valuable for researchers. A novel multiplex immunoassay was developed containing both mouse-specific and human-specific antibody pairs, utilizing a rigorous screening protocol specifically designed for the selection of effective human and mouse-specific capture antibodies. Specificity was confirmed by testing individual recombinant proteins, ensuring that mouse serum and plasma samples were detected exclusively by mouse-analyte capture beads, while human-analyte capture beads showed no reactivity. Using this novel multiplex immunoassay, we evaluated the safety and efficacy of IL-18 secreting CAR-T cells targeting U5 snRNP200, a ribonucleoprotein that is specifically expressed on the surface of AML cells in a syngeneic mouse model. Firstly, IL-18 secreting CAR-T-cells in the recipient mice showed both prolonged survival and marked proliferative ability. Cytokine profiling of serum collected 14 days post-transplant revealed elevated levels of murine IL-18, IFN-gamma, CXCL9, and CXCL10 while corresponding human cytokines remained undetected, confirming mouse specificity and validation of our model. This dataset establishes a baseline for future humanized mouse experiments, demonstrating the kit's utility in validating mouse models before transitioning to human applications. Citation Format: Anthony J. Saporita, Takeshi Fujino, Tina Raeber, Sarah Yoo, Brooke Gilliam, Shane Curran, Rick Wiese, Omar Abdel-Wahab, Anthony F. Daniyan. Evaluating U5 snRNP200-targeting CAR-T therapy for acute myeloid leukemia: A novel multiplex immunoassay for analyte discrimination in mouse 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 LB165.