title: Antibody Distance from the Cell Membrane Regulates Antibody Effector Mechanisms.
abstract: Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents.
authors.affiliation: Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: Kirstie L S
authors.initials: KLS
authors.lastname: Cleary
authors.affiliation: Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: H T Claude
authors.identifier: 0000-0003-0530-9480
authors.initials: HTC
authors.lastname: Chan
authors.affiliation: Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: Sonja
authors.initials: S
authors.lastname: James
authors.affiliation: Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Glennie
authors.affiliation: Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom msc@soton.ac.uk.
authors.forename: Mark S
authors.identifier: 0000-0003-2077-089X
authors.initials: MS
authors.lastname: Cragg
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D018951:Antigens, CD20 / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D000074301:CD52 Antigen; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology; D006801:Humans; D007167:Immunotherapy; D051379:Mice; D010587:Phagocytosis; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D015703:Antigens, CD; D018951:Antigens, CD20; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D000939:Epitopes; D006023:Glycoproteins; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9