An efficient strategy for cell-based antibody library selection using an integrated vector system

BMC Biotechnology

Hyerim Yoon¹  Jin Myung Song¹  Chun Jeih Ryu²  Yeon-Gu Kim¹  Eun Kyo Lee¹  Sunghyun Kang¹  Sang Jick Kim¹ 

[1] Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejon, 305-806, Republic of Korea

[2] Institute of Bioscience, Department of Bioscience and Biotechnology, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul, 143-747, Republic of Korea

关键词: scFv-Fc;    Cell panning;    CD9;    Antibody library;    Phage display;   

Others  :  1134852
DOI  :  10.1186/1472-6750-12-62

 received in 2012-06-21, accepted in 2012-09-13,  发布年份 2012

【 摘 要 】

Background

Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.

Results

A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.

Conclusions

This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

【 授权许可】

   
2012 Yoon et al.; licensee BioMed Central Ltd.

【 图 表 】

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