Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development.

通过 TNF 受体 1 (TNFR1) 的促炎轴选择性抑制肿瘤坏死因子 (TNF) 信号传导，同时不影响 TNFR2 的促生存和促进再生信号，这是一种有前途的策略，可以规避 TNF 作用完全抑制的局限性。批准的抗TNF药物。先前开发的人源化拮抗 TNFR1 特异性抗体 ATROSAB 显示出对 TNFR1 介导的细胞反应的有效抑制。由于亲代小鼠抗体 H398 具有更强的抑制潜力，我们仔细检查了两个分子的特异性结合参数，发现 ATROSAB 比 H398 解离更快。应用亲和力成熟和人源化可变结构域的重新设计，我们生成了 ATROSAB 的单价 Fab 衍生物 (13.7)，它表现出与 TNFR1 的结合增加和对 TNF 介导的 TNFR1 激活的卓越抑制，同时即使存在交叉也缺乏任何激动活性。 -连接抗体。为了改善其药代动力学特性，产生了几种 Fab13.7 衍生分子，包括聚乙二醇化 Fab、小鼠血清白蛋白融合蛋白、具有二聚化缺陷 Fc 的半 IgG 以及新设计的 Fv-Fc 格式，采用旋钮入孔技术。在这些衍生物中，Fv13.7-Fc 显示出改善的药代动力学特性和拮抗活性的最佳组合，因此代表了进一步临床开发的有希望的候选者。