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Updated: Jul 8, 2025
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
A disruptive clickable antibody design for the generation of antibody-drug conjugates
Nathanaël Rakotoarinoro1,2, Yan F K Dyck2, Simon K Krebs1,3
1Institute for Cell Therapy and Immunology branch Bioanalytics and Bioprocesses, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 14476 Potsdam-Golm, Germany.
Background:
Antibody-drug conjugates are cancer therapeutics that combine specificity and toxicity. A highly cytotoxic drug is covalently attached to an antibody that directs it to cancer cells. The conjugation of the drug-linker to the antibody is a key point in research and development as well as in industrial production. The consensus is to conjugate the drug to a surface-exposed part of the antibody to ensure maximum conjugation efficiency. However, the hydrophobic nature of the majority of drugs used in antibody-drug conjugates leads to an increased hydrophobicity of the generated antibody-drug conjugates, resulting in higher liver clearance and decreased stability.
Methods:
In contrast, we describe a non-conventional approach in which the drug is conjugated in a buried part of the antibody. To achieve this, a ready-to-click antibody design was created in which an azido-based non-canonical amino acid is introduced within the Fab cavity during antibody synthesis using nonsense suppression technology. The Fab cavity was preferred over the Fc cavity to circumvent issues related to cleavage of the IgG1 lower hinge region in the tumor microenvironment.
Results:
This antibody design significantly increased the hydrophilicity of the generated antibody-drug conjugates compared to the current best-in-class designs based on non-canonical amino acids, while conjugation efficiency and functionality were maintained. The robustness of this native shielding effect and the versatility of this approach were also investigated.
Conclusions:
This pioneer design may become a starting point for the improvement of antibody-drug conjugates and an option to consider for protecting drugs and linkers from unspecific interactions.
Insights
This study introduces a novel method for antibody-drug conjugates (ADCs) by conjugating cytotoxic drugs to a buried antibody site. This approach enhances ADC hydrophilicity and stability, improving cancer therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Antibody-drug conjugates (ADCs) are targeted cancer therapeutics combining antibody specificity with drug toxicity.
- Current ADC strategies conjugate drugs to surface-exposed antibody regions, often leading to increased hydrophobicity, liver clearance, and decreased stability due to drug-linker properties.
- Optimizing ADC design is crucial for improving therapeutic efficacy and safety profiles.
Purpose of the Study:
- To develop a novel antibody-drug conjugate (ADC) design strategy that improves hydrophilicity and stability.
- To explore a non-conventional drug conjugation approach by targeting buried antibody sites.
- To maintain conjugation efficiency and therapeutic functionality while mitigating hydrophobicity-related issues.
Main Methods:
- Engineered a "ready-to-click" antibody design incorporating an azido-based non-canonical amino acid within the Fab cavity using nonsense suppression technology.
- Introduced the drug-linker conjugation site into a buried region of the antibody, specifically the Fab cavity, to avoid Fc-related cleavage issues.
- Investigated the impact of this buried conjugation strategy on ADC hydrophilicity, stability, and functionality.
Main Results:
- The novel antibody design significantly increased the hydrophilicity of the resulting antibody-drug conjugates compared to existing non-canonical amino acid-based ADCs.
- Conjugation efficiency and the overall functionality of the ADCs were successfully maintained.
- The study demonstrated the robustness of the "native shielding" effect and the versatility of the proposed conjugation approach.
Conclusions:
- This pioneering antibody-drug conjugate design offers a new strategy for improving ADC properties by enhancing hydrophilicity and stability.
- The approach provides a potential method for protecting drugs and linkers from non-specific interactions, reducing off-target effects.
- This design represents a significant advancement and a valuable option for future ADC development in cancer therapy.
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