Antibody Engineering Using Phage Display with a Coiled-Coil Heterodimeric Fv Antibody Fragment

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to VH and VL for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of VH frameworks and VH-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering

Affinity of VH variants at 35°C.

<p>Affinity of VH variants at 35°C.</p

Affinity of variants post humanization or maturation at 25°C.

<p>Affinity of variants post humanization or maturation at 25°C.</p

An overview of the coiled-coil Fv (ccFv) construct.

<p>(A) The ccFv molecule design. The V_H and V_L are heterodimerized through the interaction of GR1 and GR2 coiled-coil domains. (B) The amino acid sequences of GR1, GR2, linker, and spacer. (C) pABMD5, the ccFv phage display vector. (D) pABMX168, the ccFv expression vector in <i>Pichia</i>.</p

The functional phage display of the ccFv fragment.

<p>(A) Phage ELISA was conducted using the phages displaying either the ccFv or scFv fragment encoding the same anti-VEGF antibody. The results demonstrated that ccFv was functionally assembled, expressed, and displayed on phages. The display level was higher by almost one order of magnitude than scFv. (B) A western blot of phages displaying ccFv and scFv fragments. The same amounts of phage particles displaying either the ccFv or scFv fragment were analyzed by SDS-PAGE and anti-HA western blotting. More intact ccFv-pIII fusion proteins were detected than the intact scFv-pIII fusion proteins, suggesting a higher ccFv phage display level.</p

The framework amino acid sequences of the leads selected from the ccFv humanization library for the anti-VEGF antibody.

<p>All three leads, X72, X76, and X78, differed from WT at the E6Q and L11V mutations in the V_H framework 1. The Kabat numbering scheme was used for residue numbering.</p