title: Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv.
abstract: Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3epsilongamma recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.
authors.affiliation: Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 10 Rm 3D46, 10 Center Drive, Bethesda, Maryland 20892-1216, USA.
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
authors.forename: Geun-Bae
authors.initials: GB
authors.lastname: Kim
authors.forename: Jung-Hee
authors.initials: JH
authors.lastname: Woo
authors.forename: Yuan Yi
authors.initials: YY
authors.lastname: Liu
authors.forename: Askale
authors.initials: A
authors.lastname: Mathias
authors.forename: Scott
authors.initials: S
authors.lastname: Stavrou
authors.forename: David M
authors.initials: DM
authors.lastname: Neville
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000235:genetics* / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics; D019281:Dimerization; D004167:Diphtheria Toxin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000882:Haplorhini / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016296:Mutagenesis; D009154:Mutation; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012204:Rh-Hr Blood-Group System / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology
chemical_list: D017252:CD3 Complex; C452842:CD3 antigen, gamma chain; D003062:Codon; D004167:Diphtheria Toxin; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System