Jhajj, Schardt, and Khalasawi et al. report a systematic method for discovering single-chain antibodies with unique epitopes (relative to clinical-stage antibodies) targeting TNF receptors, which were then fused to the light chains of the clinical-stage IgGs to generate human biepitopic agonist antibodies, eliminating the need for animal immunization, humanization, and molecular reformatting. Biepitopic OX40 antibodies showed potent, FcγR-independent primary human CD4+ T cell activation. The competition-based antibody screening platform was generalizable to other members of the TNF receptor superfamily (CD137; 41-BB).
Contributed by Shishir Pant
ABSTRACT: 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.
Author Info: (1) Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (2) Departmen
Author Info: (1) Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (2) Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (3) Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (4) Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (5) Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (6) Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (7) Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (8) Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. (9) Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.
