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Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Published on: September 14, 2018
Single Mutation on Trastuzumab Modulates the Stability of Antibody-Drug Conjugates Built Using Acetal-Based Linkers
Xhenti Ferhati1, Ester Jiménez-Moreno1, Emily A Hoyt2
1Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, Spain.
Abstract:
Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.
Insights
Antibody-drug conjugates (ADCs) stability is crucial for cancer therapy. Researchers found that the antibody's chemical environment, specifically Lys-207 in trastuzumab, significantly impacts ADC linker stability and drug release.
Area of Science:
- Bioconjugation Chemistry
- Antibody-Drug Conjugate Therapeutics
- Protein Engineering
Background:
- Antibody-drug conjugates (ADCs) are targeted therapies for cancer treatment.
- ADC stability in circulation and targeted payload release are critical for efficacy and safety.
- Understanding factors influencing ADC stability at the atomic level is essential for developing improved therapeutics.
Purpose of the Study:
- To investigate how the site and microenvironment of the trastuzumab antibody affect ADC conjugation and linker stability.
- To elucidate the role of specific amino acid residues in trastuzumab on linker hydrolysis and ADC stability.
- To provide insights for designing more stable and effective ADCs.
Main Methods:
- Utilized a combination of synthesis, bioconjugation, linker technology, and site-directed mutagenesis.
- Employed computational modeling to study the influence of the antibody's microenvironment.
- Investigated trastuzumab variants, including thiomab variants, and modified conjugation sites.
Main Results:
- The chemical environment of trastuzumab's conjugation site significantly impacts the stability of acid-sensitive linkers (acetals).
- Lys-207 residue near Cys-205 can act as an acid catalyst, promoting acetal hydrolysis and stabilizing ADCs.
- Mutating Lys-207 or using longer linkers enhanced conjugate stability by preventing undesired reactions.
Conclusions:
- The microenvironment of antibody conjugation sites is a key determinant of ADC stability.
- Strategic modification of antibody residues, like Lys-207, can optimize linker stability and ADC performance.
- This research offers valuable insights for the rational design of next-generation antibody-drug conjugates.
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