title: Anti-vascular endothelial growth factor receptor (VEGFR) 2 autoantibody identification in glioblastoma patient using single B cell-based antibody gene cloning.
abstract: Antibody direct cloning from single B cells is simple and efficient and has been successful in antibody identification of infectious diseases. However, although a recent whole-exome sequencing revealed abundant heterogeneic mutation accumulation in cancers, identification and synthesis of autoantibodies against specific cancer-associated antigens is still difficult in cancer patients owing to the very small number of B cells producing autoantibodies. In the present study, to identify autoantibodies targeting tumor antigens, we measured the titer of autoantibodies in high-grade glioma patients' plasma and identified two patients with elevated autoantibodies to a few transmembrane proteins. Specific B cells producing autoantibody against vascular endothelial growth factor receptor (VEGFR) 2 were immunostained with labeled protein and anti-human IgG antibody, and then collected by a single cell sorter. Finally, 22 antibody genes were successfully identified using direct IgG cloning from single B cell mRNA, and two antibody clones were found to have significant VEGFR2-specific binding affinity. The current direct human IgG gene cloning technique for identifying human antibodies derived from IgG-memory B cells avoids time-consuming procedures such as phage display-based antibody-library screening, and therefore may be applicable to identifying human autoantibodies in a variety of disorders including cancers even when antibody elevation is not detected because of a very small number of memory B cells.
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Iizuka
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Komiyama
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Oshita
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kume
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ashizawa
authors.affiliation: Department of Neurosurgery, Shizuoka Cancer Center Hospital, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Mitsuya
authors.affiliation: Department of Neurosurgery, Shizuoka Cancer Center Hospital, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan.
authors.forename: Nakamasa
authors.initials: N
authors.lastname: Hayashi
authors.affiliation: Department of Neurosurgery, Shizuoka Cancer Center Hospital, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nakasu
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Japan. Electronic address: y.akiyama@scchr.jp.
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Akiyama
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000096:biosynthesis* / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D001932:Brain Neoplasms / Q000235:genetics / Q000276:immunology / Q000473:pathology / Q000628:therapy; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D015723:Gene Library; D005909:Glioblastoma / Q000235:genetics / Q000276:immunology / Q000473:pathology / Q000628:therapy; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007156:Immunologic Memory; D007167:Immunotherapy / Q000379:methods; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology*; D059010:Single-Cell Analysis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D001323:Autoantibodies; D007074:Immunoglobulin G; D012333:RNA, Messenger; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Direct antibody gene cloning; Human IgG ELISA; Human monoclonal antibody; Single B cell; VEGFR2