One-Step Assembly of Homogeneous DAR4 ADCs via a Traceless Fc Ligand-Directed Acylation Strategy

Area of Science:

• Bioconjugation Chemistry
• Antibody-Drug Conjugate (ADC) Development
• Protein Engineering

Background:

• Site-specific antibody-drug conjugates (ADCs) offer improved pharmacological properties.
• Achieving homogeneous ADCs with controlled drug-to-antibody ratios (DAR) is crucial for therapeutic efficacy.
• Hydrophobicity-driven aggregation is a common challenge in DAR4 ADCs.

Purpose of the Study:

• To develop a novel one-step strategy for producing homogeneous ADCs with a DAR of 4.
• To optimize conjugation conditions to mitigate aggregation and maintain efficiency.
• To evaluate the therapeutic potential and safety profile of the resulting ADCs.

Main Methods:

• Utilized a Fc ligand-directed thioester-based acylating reagent for conjugation.
• Employed a β-glutamic acid-based branching linker for precise attachment of four MMAE molecules per antibody.
• Systematically optimized buffer composition and pH to control aggregation and conjugation efficiency.
• Validated the method across multiple IgG subtypes and target antigens (HER2, cMet, ROR1, FRα).

Main Results:

• Successfully prepared homogeneous ADCs with a DAR of 4 in a single step.
• Mitigated aggregation issues common in K248-linked DAR4 ADCs through buffer and pH optimization.
• Demonstrated high conjugation efficiency and tolerance to varying protein concentrations.
• Developed ADC-4 with enhanced aggregation stability, potent tumor suppression, and a favorable safety profile.

Conclusions:

• The novel one-step strategy enables efficient and precise synthesis of homogeneous DAR4 ADCs.
• Optimized conditions overcome aggregation challenges, leading to stable and effective biotherapeutics.
• The developed ADCs, particularly ADC-4, show significant promise for cancer therapy due to their efficacy and safety.