Christian Doppler Laboratory for Innovative Immunotherapeutics, Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.
Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.
Biochim Biophys Acta Gen Subj. 2022 Jul;1866(7):130155. doi: 10.1016/j.bbagen.2022.130155. Epub 2022 Apr 22.
Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation.
We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the C2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines.
Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines.
By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs.
Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.
毒素实体与抗体的位点特异性偶联已成为抗体药物偶联物（ADC）合成的一种流行方法，因为它可得到均一的产物，并允许自由选择合适的偶联位点。
我们在两种模型抗体（西妥昔单抗和曲妥珠单抗）的 C2 结构域的 FG 环 N 端引入了一个短的基序，其中包含一个被芳香族残基包围的单个半胱氨酸。用疏水相互作用色谱法和质谱法检查与毒性有效负载的缀合程度，并在抗原过表达的细胞系上测试所得缀合物的活性。
抗体突变体可与马来酰亚胺基连接子快速偶联，并赋予毒素有效负载，而修饰并未损害其与靶细胞系的反应性或对其生物物理性质产生负面影响。未经任何预先还原，多达 50%的抗体制剂被发现与每个分子结合两个毒素。在用制备型疏水相互作用色谱法分离该馏分后，ADC 可在靶细胞系上引起强烈的细胞毒性作用。
通过微调反应性半胱氨酸残基的微环境，该策略为生产位点选择性偶联 ADC 提供了简化的方案。
我们独特的方法允许生成具有受控化学组成的治疗性 ADC，从而便于优化其药理活性。这种用于定向偶联的策略将来可能会简化使用正交技术引入双有效负载（“双弹头”）的 ADC 的构建。
