title: The influence of antibody fragment format on phage display based affinity maturation of IgG.
abstract: Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly,single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs.Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.
authors.forename: Miriam
authors.initials: M
authors.lastname: Steinwand
authors.forename: Patrick
authors.initials: P
authors.lastname: Droste
authors.forename: André
authors.initials: A
authors.lastname: Frenzel
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
mesh_terms: D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017730:Ki-1 Antigen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D017730:Ki-1 Antigen; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies