ADC High-throughput
Antibody Conjugation

The mainstream method currently used is cysteine conjugation. By adding a reducing agent, the interchain disulfide bonds of the antibody are reduced at a specific temperature to generate free thiol groups. Subsequently, payload with a linker is added, and the maleimide on the linker reacts with the thiol group through Michael addition, thereby conjugating the small-molecule drug to the antibody.

• (1)Antigen Binding: ADCs circulate in the bloodstream, finding and binding to their cellular targets. Non-specific binding can lead to off-target toxicity.

• (2)Endocytosis: The mAb component of the ADC binds to FcRn, which is mainly expressed in endosomes. The ADC is then recycled back to the cell surface and exposed to physiological pH, releasing it from FcRn and extending its half-life in vivo.

• (3)Lysosomal Degradation: For ADCs with cleavable linkers, the cleavage mechanism(e.g., hydrolysis, protease-mediated cleavage, or reductive cleavage of disulfide bonds) occurs in early or late endosomes without progressing to lysosomal transport. For ADCs with non-cleavable linkers, the proton pumps on lysosomes create an acid-unstable environment that promotes ADC degradation and protease-mediated hydrolysis (e.g., by cathepsin B and plasmin).

• (4)Payload Release: The ADC undergoes catabolism and releases the toxic payload, which is then transported from the lysosomal lumen to the cytoplasm.

• (5)Target Cell Apoptosis: The cytotoxic drug induces cell apoptosis or other forms of cell death by inter