Evaluation of ketone-oxime method for developing therapeutic on-demand cleavable immunoconjugates

Area of Science:

• Bioconjugation Chemistry
• Immunology
• Cancer Therapeutics

Background:

• Antibody molecules are crucial in medical science for diagnostics and therapeutics, particularly in cancer therapy where monoclonal antibodies (mAbs) deliver payloads to cancer cells.
• Current limitations exist in efficiently conjugating diverse targeting molecules and peptidomimetics to antibodies.
• Novel conjugation strategies are essential for advancing antibody-based medical applications.

Purpose of the Study:

• To develop and optimize a novel keto-oxime conjugation method for attaching protease-susceptible linkers to antibodies.
• To enable the conjugation of a variety of functional moieties to antibodies for targeted delivery and controlled release applications.
• To validate the method's efficiency, stability, and compatibility with different functional groups.

Main Methods:

• A two-step keto-oxime conjugation process was employed: methyl ketone linker introduction to antibody primary amines, followed by conjugation to an aminoxy functional group.
• The method was optimized for antibody conjugation efficiency, with approximately 10 functional moieties attached per antibody.
• Conjugation was confirmed using MALDI-TOF MS and Western blot analysis; immune reactivity was preserved under acidic pH conditions.

Main Results:

• The optimized keto-oxime method successfully conjugated functional moieties to antibodies with high efficiency (approx. 10 moieties/Ab).
• MALDI-TOF MS and Western blot analysis confirmed successful conjugation without compromising antibody immune reactivity.
• In vitro protease susceptibility assays validated the method for prodrug release and clearance of excess conjugates.

Conclusions:

• The developed keto-oxime conjugation method provides an efficient and orthogonal approach for antibody modification.
• This method is suitable for creating antibody-drug conjugates (ADCs) and other targeted therapeutic agents.
• The technique's compatibility with various functional groups and its utility in prodrug release assays offer significant potential in cancer therapy and beyond.