Yu et al. screened nine anti-CD40 monoclonal antibodies (mAbs), and found that the mAbs that bind the part of CD40 farthest away from the cell membrane do not compete with CD40L ligation and act as agonists, while those binding closer to the membrane interfere with CD40L binding and act as antagonists. For most, but not all, mAbs, agonism also depended on affinity for FcγRIIB, accessibility of mAb Fc (based on epitope location), and local levels of FcγRIIB expression.

Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.

Author Info: (1) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (2) Antibody and Vaccine Group, Cancer Sciences Unit,

Author Info: (1) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (2) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (3) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (4) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (5) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (6) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (7) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (8) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (9) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (10) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (11) Protein Core Facility, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (12) Protein Core Facility, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (13) Hamburg Centre for Ultrafast Imaging & Institute for Nanostructure and Solid State Physics, University of Hamburg, 20146 Hamburg, Germany. (14) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK; Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK. (15) Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK; Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK. (16) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. (17) Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton SO16 6YD, UK. Electronic address: ann.white@ucb.com.