title: Design and screening of a chimeric survivin-specific nanobody and its anticancer activities in vitro.
abstract: Survivin is a strong inhibitor of apoptosis protein and a promising target for cancer prevention and treatment. Here, we report the design and preparation of novel chimeric nanobodies (Nbs) that could specifically bind to survivin. We screened the peptides from phage-displayed libraries (7-mer, 12-mer) for nonconserved sequences of complementarity-determining regions (CDRs) in the scaffold of the Nb. By a combination of the nonconserved sequences for CDRs, the corresponding chimeric Nbs (10 Nbs) were prepared with genetic operations. The antisurvivin Nb TAT-Nb4A (a fusion with cellular transduction peptide TAT) was found to be the most efficient antibody on the basis of the results from enzyme-linked immunosorbent assay, MTT, and flow cytometry when these nanobodies were tested with hepatoma carcinoma cell HepG2. TAT-Nb4A could inhibit the growth of HepG2 and promote cancer cell apoptosis significantly in a dose-dependent and time-dependent manner: the apoptosis rate reached 52.5% when the concentration of TAT-Nb4A was 120 μg/ml. Western blotting with cells expressing survivin showed that the prepared nanobody could efficiently bind to expressed survivin and blocked the signaling pathway in which survivin played a role. This study provided a convenient and feasible method of obtaining a novel specific Nb with the case of survivin as a good example.
authors.affiliation: aSchool of Biotechnology and State Key Laboratory of Bioreactor Engineering bSchool of Pharmacy and Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.forename: Hua
authors.initials: H
authors.lastname: Guo
authors.forename: Wenyun
authors.initials: W
authors.lastname: Zheng
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wang
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Ma
mesh_terms: D000918:Antibody Specificity; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D056945:Hep G2 Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D000077022:Survivin
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D009842:Oligopeptides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D000077022:Survivin