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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Affinity of VH variants at 35°C.

<p>Affinity of VH variants at 35°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