Systematic Evaluation of Maleimide Spacer Impact on Drug-Linker Deconjugation in Antibody-Drug Conjugates Ex Vivo and...

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Antibody-drug conjugates (ADCs) are crucial targeted therapeutics.
• Thiol-maleimide ADCs face instability issues, primarily drug-linker deconjugation and maleimide hydrolysis.
• Existing analytical methods require enhancement for comprehensive stability characterization.

Purpose of the Study:

• To develop a novel immunocapture method for ADC recovery from biological matrices.
• To quantify deconjugation and succinimide ring-opening in thiol-maleimide ADCs.
• To evaluate the impact of spacer structure on ADC stability in vivo and ex vivo.

Main Methods:

• Development of a generic immunocapture method coupled with RPLC-MS.
• Systematic evaluation of 22 thiol-maleimide ADCs in rat, mouse, and monkey sera.
• Comparison of IP-MS results with orthogonal ELISA pharmacokinetic (PK) profiles.

Main Results:

• Hydrophilic maleimido-triethylene glycol (MT) spacers showed higher ring-opening and stabilization, leading to less deconjugation than hydrophobic maleimidocaproyl (MC) spacers.
• MC spacer ADCs exhibited moderate ring-opening and substantial deconjugation in vivo.
• Spacer structure significantly influences water accessibility to the maleimide ring, impacting ADC stability regardless of antibody or drug-linker.

Conclusions:

• Spacer structure is a critical determinant of thiol-maleimide ADC stability.
• The developed IP-MS method offers sensitive and quantitative deconjugation analysis.
• Findings guide the design of more stable and effective ADCs.