Department of Biomolecular Chemistry, Institute for Molecules and Materials , Radboud University Nijmegen 6525 AJ , The Netherlands.
Department of Rheumatology , Leiden University Medical Center , Leiden , The Netherlands.
Mol Pharm. 2018 Dec 3;15(12):5565-5573. doi: 10.1021/acs.molpharmaceut.8b00741. Epub 2018 Oct 26.
Autoreactive B cells are thought to play a pivotal role in many autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease affecting ∼1% of the Western population and is hallmarked by the presence of anticitrullinated proteins antibodies (ACPA) produced by autoreactive B cells. We intend to develop a method to target and selectively eliminate these autoreactive B cells using a sequential antigen prodrug targeting strategy. As ACPA-expressing B cells are thought to play essential roles in RA-disease pathogenesis, we used this B cell response as a prototype to analyze the feasibility to generate a construct consisting of a biologically silenced, that is, blocked, antigen connected to a cytotoxic prodrug. Blocking of the antigen is considered relevant as it is anticipated that circulating autoantibodies will otherwise clear the antigen-prodrug before it can reach the target cell. The antigen-prodrug can only bind to the autoantigen-specific B cell receptor (BCR) upon enzymatic removal of the blocking group in close proximity of the B cell surface. BCR binding ultimately induces antigen-specific cytotoxicity after internalization of the antigen. We have synthesized a cyclic citrullinated peptide (CCP) antigen suitable for BCR binding and demonstrated that binding by ACPA was impaired upon introduction of a carboxy- p-nitrobenzyl (CNBz) blocking group at the side chain of the citrulline residue. Enzymatic removal of the CNBz moiety by nitroreductase fully restored citrulline-specific recognition by both ACPA and ACPA-expressing B cells and showed targeted cell death of CCP-recognizing B cells only. These results mark an important step toward antigen-specific B cell targeting in general and more specifically in RA, as successful blocking and activation of citrullinated antigens forms the basis for subsequent use of such construct as a prodrug in the context of autoimmune diseases.
自身反应性 B 细胞被认为在许多自身免疫性疾病中发挥关键作用。类风湿关节炎（RA）是一种影响约 1%的西方人群的自身免疫性疾病，其特征是存在由自身反应性 B 细胞产生的抗瓜氨酸化蛋白抗体（ACPA）。我们旨在开发一种使用顺序抗原前药靶向策略来靶向和选择性消除这些自身反应性 B 细胞的方法。由于 ACPA 表达的 B 细胞被认为在 RA 疾病发病机制中发挥重要作用，我们使用这种 B 细胞反应作为原型来分析产生由生物沉默的，即阻断的，连接到细胞毒性前药的抗原构建体的可行性。阻断抗原被认为是相关的，因为预期循环自身抗体在到达靶细胞之前会清除抗原-前药。只有在靠近 B 细胞表面的位置通过酶去除阻断基团，抗原-前药才能与抗原特异性 B 细胞受体（BCR）结合。BCR 结合最终在抗原内化后诱导抗原特异性细胞毒性。我们已经合成了适用于 BCR 结合的环状瓜氨酸肽（CCP）抗原，并证明在瓜氨酸残基的侧链上引入羧基-对硝基苄基（CNBz）阻断基团后，ACPA 的结合受到损害。硝基还原酶对 CNBz 部分的酶去除完全恢复了 ACPA 和 ACPA 表达的 B 细胞对瓜氨酸的特异性识别，并仅显示了 CCP 识别的 B 细胞的靶向细胞死亡。这些结果标志着朝着一般的抗原特异性 B 细胞靶向迈出了重要的一步，更具体地说，在 RA 中，成功的瓜氨酸化抗原阻断和激活为随后在自身免疫性疾病中使用此类构建体作为前药奠定了基础。
