Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a beta-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5'-end of the beta-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 x 10(9), greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.

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Sandwich ELISA experiment requires two mutually compatible affinity reagents, typically antibodies, that are highly sensitive and specific to the target analyte in its native conformation, and whose epitopes do not overlap each other's. Finding or developing a pair of antibodies that meet these requirements can be a challenge and many otherwise useful antibodies fail in sandwich ELISA-format analysis. In order to discover sandwich immunoassay-compatible antibodies for epidermal growth factor receptor (EGFR), we first immobilized cetuximab, a high-affinity monoclonal antibody against EGFR, on a plastic surface and affinity-captured EGFR in A431 cell lysate. Panning of a phage antibody library on the surface-captured antigen yielded a couple of antibody clones that can be paired with cetuximab and also with each other in the sandwich-format immunoassay. This provides a potentially useful strategy for the development of sandwich ELISA reagents or antibodies against antigens that are hard to purify. © KSBB hÉóïçêÇëW= p~åÇïáÅÜ= bifp^I= éÜ~ÖÉ= Çáëéä~óI=~åíáÄçÇóI= ÉéáÇÉêã~ä= ÖêçïíÜ= Ñ~Åíçê= êÉÅÉéíçê= EbdcoFI= Å~éíìêÉJë~åÇïáÅÜ= é~ååáåÖ= = = = =

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been used for cancer treatment. However, VEGF inhibitors alone can promote tumor invasion and metastasis by increase of intratumoral hypoxia in some preclinical models. Emerging reports regarding angiogenesis suggest Delta-like-ligand 4 (Dll4) is a promising target to augment the effects of VEGF inhibitors. Dll4 blockade inhibits tumor progression by increasing nonproductive angiogenesis whereas VEGF blockade inhibits tumor progression by decreasing angiogenesis. To evaluate the effect by inhibiting both VEGF and Dll4 on tumor angiogenesis, we have developed HD105, a novel bispecific antibody targeting VEGF and Dll4, as the format of VEGF-binding IgG linked with Dll4-binding single-chain Fv. The bispecific antibody showed potent binding affinity against VEGF (KD: 3.4 x 10-12 M) and Dll4 (KD: 4.2 x 10-8 M), which are comparable with KD values of anti-VEGF antibody (9.8 x 10-10 M) and Dll4 single targeting antibody (5.5 x 10-9 M). The HD105 bispecific antibody also inhibited interaction of Dll4 and its receptor, Notch 1 (IC50: 1 nM ± 0.2). By using in vitro cell-based assays, the HD105 effectively blocked Dll4 as well as VEGF signaling pathways in endothelial cells, resulting in downregulation of Notch intracellular domain (NICD) and VEGFR-2/extracellular-signal-regulated kinase (ERK) phosphorylation. In addition, the HD105 more efficiently inhibited tumor progression than each single-targeting antibody in A549 human lung cancer and SCH human gastric cancer xenograft models. In conclusion, the novel bispecific antibody could be potentially developed as a clinically more efficacious therapeutic antibody. Citation Format: Weon-Kyoo You, Dongheon Lee, Yu Bin Choi, Kyung Jae Kang, Dong In Kim, Jin-Hyung Ahn, Jinwon Jung, Eun-Sil Sung, Jun Seo Goo, Hyun Sook Jang, Kyung Duk Moon, Sang Hooon Lee. A novel anticancer bispecific antibody targeting VEGF and Dll4 inhibits tumor progression in lung and gastric cancer xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-97. doi:10.1158/1538-7445.AM2014-LB-97