title: Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro.
abstract: The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.
authors.affiliation: From the Department of Biologic Optimization, Thousand Oaks, California 91320. Electronic address: ling.liu@amgen.com.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: From the Department of Biologic Optimization, Thousand Oaks, California 91320.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Jacobsen
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Nancy
authors.initials: N
authors.lastname: Everds
authors.affiliation: Department of Clinical Immunology, Thousand Oaks, California 91320.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: Department of Clinical Immunology, Thousand Oaks, California 91320.
authors.forename: Yan Bin
authors.initials: YB
authors.lastname: Yu
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Nianyu
authors.initials: N
authors.lastname: Li
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Darcey
authors.initials: D
authors.lastname: Clark
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Mai Phuong
authors.initials: MP
authors.lastname: Nguyen
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Madeline
authors.initials: M
authors.lastname: Fort
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Padma
authors.initials: P
authors.lastname: Narayanan
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Kei
authors.initials: K
authors.lastname: Kim
authors.affiliation: Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Riki
authors.initials: R
authors.lastname: Stevenson
authors.affiliation: Process Development, Amgen Inc., Thousand Oaks, California 91320.
authors.forename: Linda
authors.initials: L
authors.lastname: Narhi
authors.affiliation: From the Department of Biologic Optimization, Thousand Oaks, California 91320.
authors.forename: Kannan
authors.initials: K
authors.lastname: Gunasekaran
authors.affiliation: Departments of Comparative Biology and Safety Sciences, Thousand Oaks, California 91320.
authors.forename: Jeanine L
authors.initials: JL
authors.lastname: Bussiere
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D001792:Blood Platelets / Q000276:immunology*; D045744:Cell Line, Tumor; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D008252:Macaca fascicularis; D051379:Mice; D009000:Monocytes / Q000276:immunology*; D010587:Phagocytosis / Q000187:drug effects* / Q000276:immunology; D015539:Platelet Activation / Q000187:drug effects* / Q000276:immunology; D017452:Receptors, IgG / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D017452:Receptors, IgG
keywords: Fc receptor; antibody engineering; antibody-dependent cellular cytotoxicity; complement-dependent cytotoxicity; cynomolgus macaque; immunoglobulin G (IgG); phagocytosis; platelet; platelet depletion