Jhajj Harkamal S, Schardt John S, Khalasawi Namir, Yao Emily L, Lwo Timon S, Kwon Na-Young, O'Meara Ryen L, Desai Alec A, Tessier Peter M
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.
bioRxiv. 2023 Dec 12:2023.12.11.571146. doi: 10.1101/2023.12.11.571146.
Agonist antibodies that activate cellular receptors 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 potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.
激活细胞受体的激动剂抗体正被用于从神经退行性疾病到癌症等一系列治疗应用中。对于肿瘤坏死因子（TNF）受体超家族而言，三个或更多受体的高阶聚集是其有效激活的关键。这可以通过识别同一受体上两个独特表位并介导受体超聚集的抗体来实现。然而，识别用于激活TNF受体的兼容抗体对以产生双表位抗体（也称为双特异性抗体）通常需要动物免疫，这是一个费力且不可预测的过程。在此，我们报告了一种简单的方法，可系统地识别能有效激活TNF受体的双表位抗体，而无需额外的动物免疫。我们的方法使用现成的、受体特异性IgG抗体，这些抗体缺乏内在（不依赖Fc-γ受体）的激动剂活性，首先阻断其相应表位。接下来，我们使用酵母表面展示和荧光激活细胞分选技术，从人非免疫文库中筛选能结合同一胞外域上可及表位的单链抗体。最终，将所选的单链抗体与IgG的轻链融合，生成能结合两个不同受体表位并介导有效受体激活的人四价抗体。我们通过将几种现有的针对TNF受体的临床阶段抗体，包括针对OX40的依武昔单抗和波加珠单抗以及针对CD137的乌托美单抗，转化为具有高效激动剂活性的双表位抗体，突出了该方法的广泛实用性。我们期望这种广泛可用的方法可用于系统地生成双表位抗体，以激活TNF受体超家族中的其他受体以及许多其他其激活依赖于强受体聚集的受体。
