Abstract
Antibody-drug conjugates (ADCs) rely on antibody-mediated internalization to deliver cytotoxic payloads into tumor cells. Therefore, quantitative assessment of antibody internalization is essential for ADC development, particularly during early antibody screening stages. However, conventional internalization assays, whether direct or indirect, often face challenges such as low throughput, reduced sensitivity, and limited target specificity due to spatial hindrance. Here, we introduce a versatile 3C peptide conjugate platform that utilizes the high-affinity binding of IgG by the C1-C3 domains of streptococcal protein G. This platform includes 3C-toxin for cytotoxicity-based internalization detection and 3C-pHAb for pH-sensitive fluorescent tracking. By simply incubating these reagents with antibodies, effective labeling is achieved without complex modifications, enabling sensitive and high-throughput evaluation of internalization. We validated the platform across multiple tumor-associated targets, including HER2, CDH6, LIV-1, LYPD3, and GPC3, demonstrating a strong correlation between 3C-based assays and the cytotoxic efficacy of corresponding ADCs. Notably, 3C-toxin showed superior target promiscuity compared to traditional DT3C methods, expanding applicability to a broader range of antigens. This platform provides a scalable solution for antibody internalization analysis, positioned to accelerate ADC discovery by providing reliable early-stage screening metrics.