title: The Fab Fragment of a Human Anti-Siglec-9 Monoclonal Antibody Suppresses LPS-Induced Inflammatory Responses in Human Macrophages.
abstract: Sepsis is a major cause of death for hospitalized patients and is characterized by massive overreaction of immune responses to invading pathogens which is mediated by cytokines. For decades, there has been no effective treatment for sepsis. Sialic acid-binding, Ig-like lectin-9 (Siglec-9), is an immunomodulatory receptor expressed primarily on hematopoietic cells which is involved in various aspects of inflammatory responses and is a potential target for treatment of sepsis. The aim of the present study was to develop a human anti-Siglec-9 Fab fragment, which was named hS9-Fab03 and investigate its immune activity in human macrophages. We began by constructing the hS9-Fab03 prokaryotic expression vector from human antibody library and phage display. Then, we utilized a multitude of assays, including SDS-PAGE, Western blotting, ELISA, affinity, and kinetics assay to evaluate the binding affinity and specificity of hS9-Fab03. Results demonstrated that hS9-Fab03 specifically bind to Siglec-9 antigen with high affinity, and pretreatment with hS9-Fab03 could attenuate lipopolysaccharide (LPS)-induced TNF-α, IL-6, IL-1β, IL-8, and IFN-β production in human PBMC-derived macrophages, but slightly increased IL-10 production in an early time point. We also observed similar results in human THP-1-differentiated macrophages. Collectively, we prepared the hS9-Fab03 with efficient activity for blocking LPS-induced pro-inflammatory cytokines production in human macrophages. These results indicated that ligation of Siglec-9 with hS9-Fab03 might be a novel anti-inflammatory therapeutic strategy for sepsis.
authors.affiliation: Department of Infectious Disease, Anhui Medical University Affiliated with Bayi Clinical College, Hefei, China; Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing, China.
authors.forename: Sasa
authors.initials: S
authors.lastname: Chu
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing, China; Department of Emergency Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Infectious Disease, Anhui Medical University Affiliated with Bayi Clinical College, Hefei, China; Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing, China.
authors.forename: Na
authors.initials: N
authors.lastname: You
authors.affiliation: Department of Emergency Medicine, Jinling Hospital, Medical School of Nanjing University , Nanjing , China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Huadong Medical Institute of Biotechniques , Nanjing , China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital , Nanjing , China.
authors.forename: Binggang
authors.initials: B
authors.lastname: Cai
authors.affiliation: Huadong Medical Institute of Biotechniques , Nanjing , China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Huadong Medical Institute of Biotechniques , Nanjing , China.
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Traditional Chinese Pharmacology, Chinese Pharmaceutical University , Nanjing , China.
authors.forename: Qiannan
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital , Nanjing , China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital , Nanjing , China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Huadong Medical Institute of Biotechniques , Nanjing , China.
authors.forename: Changjun
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Emergency Medicine, Jinling Hospital, Medical School of Nanjing University , Nanjing , China.
authors.forename: Shinan
authors.initials: S
authors.lastname: Nie
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing, China; Department of Pathology, Key Laboratory of Antibody Technique of the Ministry of Health, NJMU, Nanjing, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Infectious Disease, Anhui Medical University Affiliated with Bayi Clinical College, Hefei, China; Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing, China.
authors.forename: Maorong
authors.initials: M
authors.lastname: Wang
keywords: Fab fragment; LPS; Siglec-9; TLR4; human anti-Siglec-9 antibody; human macrophages; sepsis