Department of Biomedical Engineering, University of Utah, Salt Lake City, Utah 84112, United States.
Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84132, United States.
Bioconjug Chem. 2020 Aug 19;31(8):1960-1970. doi: 10.1021/acs.bioconjchem.0c00324. Epub 2020 Jul 20.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.
类风湿关节炎（RA）是一种自身免疫性疾病，其特征为滑膜关节的慢性炎症和蛋白酶诱导的软骨降解。目前针对 RA 的生物治疗方法可以有效减轻症状，主要通过中和促炎细胞因子 TNFα；然而，持续、无差别地过度抑制炎症因子会显著削弱宿主免疫系统，导致机会性感染并中断治疗。我们假设，通过与胶原杂交肽（CHP）缀合，将抗 TNFα 治疗药物靶向定位到关节炎关节中存在的变性胶原（dCol），将减少非靶向抗原结合并维持局部免疫抑制。我们分离了临床批准的抗 TNFα 治疗药物英夫利昔单抗（iFab）的抗原结合片段，并通过赖氨酸与 SMCC 接头的连接，制备了 iFab-CHP 缀合物。通过 LC-MS 成功缀合后，通过 ELISA 样测定法对 iFab-CHP 的结合亲和力进行了表征，结果显示其相对于英夫利昔单抗具有相当的抗原结合能力，相对于 CHP 具有相当的 dCol 结合能力，并且具有同时结合 dCol 和 TNFα 的杂交能力。我们还进一步证明了 Fab-CHP 在体内高 dCol 区域的定位，并在一项小鼠初步研究中通过组织学染色（番红 O 和 H&E）评估了其有前景的治疗效果。
