3C conjugates: a highly sensitive platform for antibody internalization assessment in ADC development

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.