Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

激动剂抗体正在寻求用于从神经退行性疾病到癌症的治疗应用。对于肿瘤坏死因子 (TNF) 受体超家族，三个或更多受体的高阶聚类是其激活的关键，这可以使用识别两个独特表位的抗体来实现。然而，双表位（即双互补位）抗体的产生通常需要动物免疫并且是费力且不可预测的。在这里，我们报告了一种简单的方法，用于识别有效激活 TNF 受体的双表位抗体，而无需额外的动物免疫。我们的方法使用现有的、缺乏内在激动剂活性的受体特异性 IgG 来阻断其相应的表位，然后选择结合可接近表位的单链抗体。选定的抗体与 IgG 的轻链融合，生成人四价抗体。我们通过将几种针对 OX40 和 CD137 (4-1BB) 的临床阶段抗体转化为具有有效激动剂活性的双表位抗体，强调了这种方法的广泛实用性。