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A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a
Marco A Pometti1,2, Giuseppe Di Natale3, Giancarlo Geremia1,4
1Nuclear Medicine Department, Cannizzaro Hospital, Via Messina 829, 95126 Catania, Italy.
Bioconjugate Chemistry
|February 17, 2024
Summary
This study introduces a regioselective bioconjugation method for creating immunoconjugates, improving antibody-drug delivery. The new approach enhances the chemical quality of antibody-drug conjugates by precisely controlling payload attachment.
Area of Science:
- Bioconjugation Chemistry
- Antibody Engineering
- Mass Spectrometry
Background:
- Immunoconjugates deliver targeted therapies by linking antibodies to cytotoxic payloads.
- Traditional lysine conjugation methods yield heterogeneous mixtures, potentially reducing efficacy.
- Site-selective bioconjugation is crucial for developing homogeneous and effective antibody-drug conjugates.
Purpose of the Study:
- To develop an intradomain regioselective bioconjugation strategy for monoclonal antibodies (mAbs).
- To characterize the site of conjugation and the resulting immunoconjugate mixture using mass spectrometry.
- To establish a domain mapping workflow for precise quantification of chelator attachment.
Main Methods:
- Kinetically controlled reaction of Trastuzumab with a DOTA-NHS ester at slightly basic pH.
- Liquid chromatography-mass spectrometry (LC-MS) for chelator-antibody ratio (CAR) and conjugation site analysis.
- Selective domain denaturation and MS-workflow for quantifying chelator distribution across mAb domains.
Main Results:
- Identified four key lysine residues (K188, K30, K293, K417) involved in conjugation across different domains.
- Achieved an average CAR of 0.9, with monoconjugation predominant on heavy chains.
- Domain mapping revealed specific chelator distribution: CH3 (13%), CH2 (12.5%), VH (11%), and CL (6%).
Conclusions:
- The developed intradomain regioselective bioconjugation offers precise control over immunoconjugate formation.
- The MS-based domain mapping workflow enables detailed characterization of conjugate heterogeneity.
- This approach enhances the chemical quality and potential therapeutic efficacy of immunoconjugates.
