Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro

• Published in: The Journal of biological chemistry Vol. 292; no. 5; pp. 1876 - 1883
• Main Authors: Liu, Ling, Jacobsen, Frederick W., Everds, Nancy, Zhuang, Yao, Yu, Yan Bin, Li, Nianyu, Clark, Darcey, Nguyen, Mai Phuong, Fort, Madeline, Narayanan, Padma, Kim, Kei, Stevenson, Riki, Narhi, Linda, Gunasekaran, Kannan, Bussiere, Jeanine L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.02.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; antibody engineering; antibody-dependent cellular cytotoxicity; Blood Platelets - immunology; Cell Line, Tumor; complement-dependent cytotoxicity; cynomolgus macaque; Fc receptor; Humans; immunoglobulin G (IgG); Immunoglobulin G - genetics; Immunoglobulin G - immunology; Immunoglobulin G - pharmacology; Immunology; Macaca fascicularis; Mice; Monocytes - immunology; phagocytosis; Phagocytosis - drug effects; Phagocytosis - immunology; platelet; Platelet Activation - drug effects; Platelet Activation - immunology; platelet depletion; Receptors, IgG - genetics; Receptors, IgG - immunology; antibody engineering; immunoglobulin G (IgG); phagocytosis; Fc receptor; antibody-dependent cellular cytotoxicity; platelet depletion; platelet; cynomolgus macaque; complement-dependent cytotoxicity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.748707

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.