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.

Phage and Yeast Display

“…The improvements in binding characteristics observed in particular Fab conformations were retained following the conversion to full-length IgG. (36). These findings suggest that the Fab format is a successful antibody fragment surrogate for the selection, prediction, and optimization of IgG candidates.…”

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes

“…These scFvs bind to the peptide-HLA complexes on the surface of cells and can kill those cells when complement is present. Converting an scFv into a complete, bivalent antibody containing the Fc region sometimes results in loss of affinity (46,47). However, we successfully generated a complete antibody using the D10 scFv sequence, and this MANAbody retained a dissociation rate constant and specificity comparable to those of the scFv (SI Appendix, Table S5 and Fig.…”

Hybridoma Technology: The Preferred Method for Monoclonal Antibody Generation for in vivo Applications

“…Since the process of hybridoma-based mAb generation takes advantage of natural CDR development, there is very limited risk of those CDRs causing an immune response. In vitro methods, such as display libraries, can generate unnatural pairings of V H and V L and cause the resulting mAb to have high immunogenicity, especially after unnatural AM and recombinant pairing with a standard IgG backbone [17]. …”