Abstract 4508: Quantitation of PD-1, PD-L1, and CTLA-4 in human melanoma using SMC® high sensitivity immunoassays

Abstract Immune checkpoint proteins such as PD-1, PD-L1, and CTLA-4 play a central role in neoplastic cell immunosurveillance. Soluble forms of these cell surface proteins are currently being evaluated as potential biomarkers in the diagnosis, prognosis, and pathogenesis of several forms of cancer, such as melanoma. For this study, we first wanted to compare human plasma samples from 12 healthy donors and 12 individuals with melanoma using MILLIPLEX® Human Immuno-Oncology Checkpoint Protein Panel 1, a 17-plex immunoassay. Three immune checkpoint proteins (CD27, CD40, and TIM-3) exhibited statistically significant increases in melanoma samples versus healthy controls, despite the small sample size. While PD-1, PD-L1, and CTLA-4 trended toward increases in the disease state, differences were below the threshold of statistical significance when assessed by MILLIPLEX®. CTLA-4, in particular, was present in low abundance in circulation, with fifty percent of healthy plasma samples near the lower limit of quantification (LLoQ) for the MILLIPLEX® assay. To achieve greater sensitivity for this critical immune checkpoint protein, we developed a novel SMC® CTLA-4 high sensitivity immunoassay. SMC® technology delivers ultrasensitive biomarker measurements for low abundant proteins in comparison to the limits of traditional sandwich ELISA immunoassays. The SMC® assays for PD-1, PD-L1 and CTLA-4 consistently achieved LLoQs of 1.74 pg/mL, 0.78 pg/mL and 0.1 pg/mL, respectively. More importantly, these ultrasensitive SMC® kits allowed quantitation of these cell surface proteins in all plasma samples tested from both healthy donors and melanoma patients. Statistical differences in CTLA-4 (p-value <0.01) and PD-1 (p-value < 0.005) were observed with the SMC® kits in the melanoma cohort compared to the healthy samples. The integration of MILLIPLEX® multiplex assays and SMC® high sensitivity assays in a comprehensive workflow enables profiling of multiple analytes and a deeper understanding of immune response dynamics by accurately measuring low abundant proteins, thereby providing a powerful non-invasive tool for evaluating disease conditions. Citation Format: Anthony J. Saporita, Joseph Hwang, Matthew Lippold, Laila Faramarzi, Raad Gitan, Wen-Rong Lie, Brooke Gilliam, Rick Wiese. Quantitation of PD-1, PD-L1, and CTLA-4 in human melanoma using SMC® high sensitivity immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4508.