Development of a Cytotoxic Antibody–Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells
Abstract
:1. Introduction
2. Results
2.1. Antibody Production and Characterization
2.2. Binding Properties of 15cl12 and the ADC Derivate
2.3. Internalization Experiments
2.4. Comparison of Binding and Internalization Properties with Other Anti-IgE Antibodies
2.5. Specific Cytotoxic Activity of the ADC
3. Discussion
4. Materials and Methods
4.1. Protein Expression, Purification, and Preparation of Toxin Conjugates
4.2. Protein Characterization
4.2.1. SDS-PAGE
4.2.2. SEC-HPLC
4.2.3. HIC
4.2.4. Mass Spectrometry Analysis
4.3. Target Antigen Binding
4.3.1. Biolayer Interferometry
4.3.2. ELISA
4.3.3. Cell Culture
4.3.4. Cell Staining
4.3.5. Competition between EMPD-Binding Antibodies 15cl12 and Quilizumab
4.4. Internalization Assays
4.4.1. Quenching of Surface Fluorescence
4.4.2. Microscopy
4.4.3. Internalization of Fcε
4.5. Cytotoxicity Experiments
4.5.1. Direct Specific Cytotoxic Activity of the ADC
4.5.2. Cytotoxicity after Cross-Linking of Anti-IgE Antibodies
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Rodak, A.; Stadlbauer, K.; Bobbili, M.R.; Smrzka, O.; Rüker, F.; Wozniak Knopp, G. Development of a Cytotoxic Antibody–Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells. Int. J. Mol. Sci. 2023, 24, 14997. https://doi.org/10.3390/ijms241914997
Rodak A, Stadlbauer K, Bobbili MR, Smrzka O, Rüker F, Wozniak Knopp G. Development of a Cytotoxic Antibody–Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells. International Journal of Molecular Sciences. 2023; 24(19):14997. https://doi.org/10.3390/ijms241914997
Chicago/Turabian StyleRodak, Aleksandra, Katharina Stadlbauer, Madhusudhan Reddy Bobbili, Oskar Smrzka, Florian Rüker, and Gordana Wozniak Knopp. 2023. "Development of a Cytotoxic Antibody–Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells" International Journal of Molecular Sciences 24, no. 19: 14997. https://doi.org/10.3390/ijms241914997