Department of Chemical Biology and Drug Discovery Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG, Utrecht, The Netherlands.
Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.
Chembiochem. 2022 Nov 4;23(21):e202200408. doi: 10.1002/cbic.202200408. Epub 2022 Sep 29.
Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi-functionalization of the widely employed carrier protein CRM with a carbohydrate-based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one-step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site-specific conjugation avoids modification of T-epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation.
抗原与载体蛋白的缀合广泛用于疫苗开发。为了开发下一代结合疫苗，我们在这里描述了一种用基于碳水化合物的抗原和免疫增强剂对广泛使用的载体蛋白 CRM 进行可控多功能化的方法。该方法基于 CRM 中二硫键的选择性还原，然后使用适当功能化的二溴吡嗪二酮进行二硫键重新桥接。有效的蛋白质修饰需要还原和二溴吡嗪二酮的功能化作为一步法进行，同时控制反应温度。此外，当使用具有 TLR7/8 激动剂和环辛炔等功能实体的二溴吡嗪二酮进行连接时，连接最成功。 定点缀合可避免载体蛋白 T-表位的修饰，并且免疫增强剂的共价连接将确保细胞因子在疫苗与相关免疫细胞相互作用的部位产生，从而有效增强免疫。
