title: A humanized single-chain variable fragment antibody against beta3 integrin in Escherichia coli.
abstract: Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique antibody (Ab) against platelet GPIIIa49-66, which is capable of inducing oxidative platelet fragmentation in the absence of complement activation. By screening a human phage antibody library with the GPIIIa49-66 peptide as bait, we have developed several humanized phage Abs, which act similarly to the parental Ab. However, the presence of a stop codon in the heavy chain of the obtained phage clones limits their expression in soluble recombinant form. To circumvent this problem, we mutated the stop codon inside clone 11 that exhibits the highest binding activity to platelet GPIIIa49-66, resulting in a soluble scFv format (named A11) in Escherichia coli Rosseta. In in vitro binding assay, A11 exhibited similar binding specificity to parental Ab at various concentrations. Moreover, A11 is able to induce oxidative platelet fragmentation by preferentially binding to activated versus resting platelets. These findings provide a proof-of-principle for the development of a novel approach to inhibit arterial thrombosis by generating a selective scFv for the lysis of platelet-rich thrombi.
authors.affiliation: Department of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China.
authors.forename: Suying
authors.initials: S
authors.lastname: Dang
authors.forename: Tao
authors.initials: T
authors.lastname: Hong
authors.forename: Bi-Sen
authors.initials: BS
authors.lastname: Ding
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology*; D000918:Antibody Specificity; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology*; D012995:Solubility
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D018388:Codon, Terminator; C061248:ITGB3 protein, human; D039661:Integrin beta3; D009861:Onium Compounds; D010446:Peptide Fragments; D019151:Peptide Library; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C106958:platelet glycoprotein (GP)IIIa 49-66; C007517:diphenyleneiodonium