3C conjugates: a highly sensitive platform for antibody internalization assessment in ADC development
Changyan Chen1,2, Yutian Zhang1,2, Yadan Wu1,2
1State Key Laboratory of Neurology and Oncology Drug Development, Simcere Pharmaceutical Group, Nanjing, China.
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.
Insights
A new 3C peptide conjugate platform enables sensitive, high-throughput antibody internalization analysis for antibody-drug conjugate (ADC) development. This versatile tool accelerates ADC discovery by providing reliable early-stage screening metrics.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Antibody-drug conjugates (ADCs) require antibody-mediated internalization for targeted payload delivery to tumor cells.
- Accurate assessment of antibody internalization is crucial for efficient ADC development and antibody screening.
- Conventional internalization assays present limitations in throughput, sensitivity, and specificity.
Purpose of the Study:
- To introduce a novel 3C peptide conjugate platform for quantitative antibody internalization assessment.
- To enable sensitive, high-throughput, and specific evaluation of antibody internalization for ADC discovery.
- To provide a scalable solution for early-stage antibody screening in ADC development.
Main Methods:
- Utilized streptococcal protein G's C1-C3 domains for high-affinity IgG binding.
- Developed 3C-toxin for cytotoxicity-based internalization detection and 3C-pHAb for pH-sensitive fluorescent tracking.
- Validated the platform across multiple tumor-associated targets (HER2, CDH6, LIV-1, LYPD3, GPC3).
Main Results:
- Demonstrated sensitive and high-throughput antibody internalization evaluation without complex antibody modification.
- Showed a strong correlation between 3C-based assays and the cytotoxic efficacy of corresponding ADCs.
- 3C-toxin exhibited superior target promiscuity compared to traditional methods, broadening antigen applicability.
Conclusions:
- The 3C peptide conjugate platform offers a versatile and scalable solution for antibody internalization analysis.
- This platform accelerates ADC discovery by providing reliable early-stage screening metrics.
- The developed assays overcome limitations of conventional methods, enhancing ADC development efficiency.
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