Abstract C110: Revealing Hidden Off-target Risks of CTLA-4: A High-Sensitivity Assessment via the MPSA-AB5000 Platform

Abstract CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) is a critical immune checkpoint target with growing clinical relevance in immuno-oncology. However, its unique biological behavior—marked by rapid endocytosis upon T cell activation—creates significant challenges for traditional off-target screening methods such as flow cytometry. In most cases, surface CTLA-4 becomes nearly undetectable, resulting in “false negative” assessments of antibody binding and underestimated cross-reactivity risks. This issue is particularly problematic for high-affinity antibodies and bispecific formats, where weak or transient signals may be entirely missed. We employed the proprietary MPSA-AB5000 platform, which integrates a wash-free reporter cell system with signal amplification, to address the limitations of conventional FACS-based screening. CTLA-4 binding affinity was assessed using Ipilimumab across two engineered CHO cell models: CHOK1-CTLA-4 (wild-type) and CHOK1-CTLA-4-Δ23 (endocytosis-deficient). Binding signals were further evaluated under native internalization conditions without pharmacological inhibition. Standard FACS assays showed significantly diminished MFI due to internalization-induced signal loss, masking the picomolar affinity of Ipilimumab for CTLA-4. In contrast, the MPSA-AB5000 system successfully detected robust and quantifiable luciferase signals, accurately capturing antibody–antigen interactions even in the context of rapid internalization. This approach revealed true binding characteristics and mitigated the risk of underestimating off-target effects. The MPSA-AB5000 platform offers a highly sensitive and reliable solution for evaluating challenging targets such as CTLA-4 and other low-signal antigens, enabling accurate off-target assessment where conventional methods often fail. This technology provides a powerful tool for early-stage biologic screening and supports the development of safer, more effective antibody therapeutics in the immune checkpoint space. Citation Format: Thomas A. Strong, Jinying Ning, Feng Hao. Revealing Hidden Off-target Risks of CTLA-4: A High-Sensitivity Assessment via the MPSA-AB5000 Platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C110.