Engineering a unique antibody for patients with RA

类风湿关节炎是最常见的慢性自身免疫性疾病之一，可导致完全关节 如果不治疗，会造成破坏和严重残疾。类风湿性关节炎无法治愈，高达50%的类风湿性关节炎患者无法治愈 肿瘤坏死因子治疗后循环Th-17/IL-17水平显著升高对抗肿瘤坏死因子治疗的反应 封锁。对于这一亚群的RA患者，一条新的途径的中断会削弱肿瘤坏死因子之间的协同作用 (M1巨噬细胞)和IL-17(Th-17细胞)级联可能解决RA治疗中的关键障碍。因此 本项目的目的是开发一种治疗类风湿关节炎患者的人TLR5抗体(Ab) 是由效应器巨噬细胞和T细胞之间的串扰驱动的。 我们证明了TLR5与其在关节中表达的天然配体的连接，转化了RA 外周血(PB)细胞转化为促炎性M1巨噬细胞，产生高水平的肿瘤坏死因子、白介素1和 IL-6。此外，由TLR5驱动的M1巨噬细胞产生的IL-6可以将原始T细胞分化为 炎性RA TH-17细胞，分泌IL-17A、IL-17F、IL-22、IL-24、IL-26、CCL20和GM-CSF。在老鼠身上， 全身和局部注射TLR5激动剂加剧关节肿胀；相反，抗TLR5抗体治疗 缓解胶原蛋白诱导的关节炎(CIA)关节炎症。 为了研究TLR5抗体作为治疗类风湿性关节炎的潜在方法的作用，我们与科学家在 Abwiz Inc.使用TLR5抗原，筛选人片段抗原结合(Fab)噬菌体展示文库 用于TLR5高亲和力粘合剂。对多达40个阳性克隆进行了测序，并选择了10个TLR5 Fab克隆 表达、纯化并用ELISA法测定TLR5结合活性和交叉反应性。10个Fab克隆是 Shahrara实验室检测了TLR5在人和小鼠细胞中的中和能力，其中一种是 基于其卓越的阻挡能力而被选中。 本项目的总体目标是开发一种用于RA治疗的抗TLR5抗体。在第一阶段，我们的方法 是提高抗TLR5抗体的亲和力，以达到商业范围内的Kd值 可用ABS。通过使用噬菌体展示抗体库的定点突变提供的候选基因将是 测试了它们在人体细胞中的TLR5阻断亲和力。随后，最有希望的候选人将是 测试它们在人源化RA中消除RA滑液促进炎症反应的能力 老鼠模型。该项目的长期目标是产生一种安全的、全新的针对RA的TLR5抗体 对目前的治疗没有反应的患者。

RA is one of the most common chronic autoimmune disorders that can lead to complete joint destruction and severe disability if untreated. There is no cure for RA and up to 50% of RA patients do not respond to anti-TNF therapies as circulating Th-17/IL-17 levels are highly elevated subsequent to TNF blockade. For this subset of RA patients, disruption of a novel pathway that impairs the synergy between TNF (M1 macrophages) and IL-17 (Th-17 cells) cascades may resolve the critical barrier in RA treatment. Hence objective of this project is to develop a therapeutic human TLR5 antibody (Ab) for RA patients whose disease is driven by the cross-talk between the effector macrophages and T cells. We documented that ligation of TLR5 to its natural ligand expressed in the joints, transforms RA peripheral blood (PB) cells into proinflammatory M1 macrophages which produce high levels of TNF, IL-1 and IL-6. In addition, IL-6 produced from TLR5 driven M1 macrophages can differentiate the naïve T cells into inflammatory RA TH-17 cells that secrete IL-17A, IL-17F, IL-22, IL-24, IL-26, CCL20 and GM-CSF. In mice, systemic and local injection of a TLR5 agonist exacerbates joint swelling; conversely anti-TLR5 Ab treatment alleviates collagen induced arthritis (CIA) joint inflammation. To investigate the role of TLR5 Ab as a potential treatment for RA, we have partnered with scientists at Abwiz Inc. Using a TLR5 antigen, a human fragment antigen-binding (Fab) phage display library was screened for TLR5 high affinity binders. Up to 40 positive clones were sequenced and 10 selected TLR5 Fab clones were expressed, purified and assessed by ELISA for TLR5 binding and cross reactivity. Ten Fab clones were examined by the Shahrara lab for TLR5 neutralization capacity in human and murine cells and one was selected based on its superior blocking capacity. The overall goal of this project is to develop an anti-TLR5 Ab for RA therapy. In Phase I, our approach is to enhance the affinity of anti-TLR5 Ab in order to reach a Kd value that is within the range of commercially available Abs. Candidates provided through site directed mutagenesis using phage-display Ab library, will be tested for their TLR5 blocking affinity in human cells. Subsequently, the most promising candidates will be tested for their ability to abrogate RA synovial fluid from promoting inflammatory response in humanized RA mouse model. The long term goal of this project is to generate a safe and completely novel TLR5 Ab for RA patients that do not respond to the current therapies.