Generation and analysis of the improved human HAL9/10 antibody phage display libraries

BMC Biotechnology

Research Article

Henk Garritsen¹  Mark Schütte²  Björn Hock²  Lars Toleikis²  Florian Tomszak³  Stefan Dübel³  Doris Meier³  Michael Hust³  Thomas Schirrmann⁴  André Frenzel⁴  Jonas Kügler⁵  Sonja Wilke⁶ 

[1] Klinikum Braunschweig g GmbH, Institut für Klinische Transfusionsmedizin, Celler Str. 38, 38114, Braunschweig, Germany;Department Vaccinology, Helmholtz-Zentrum für Infektionsforschung, Inhoffenstraße 7, 38124, Braunschweig, Germany;Merck KGaA, Darmstadt, Germany;Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany;Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany;YUMAB GmbH, Rebenring 33, 38106, Braunschweig, Germany;Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany;mAb-factory GmbH, Gelsenkirchenstr. 5, 38108, Braunschweig, Germany;mAb-factory GmbH, Gelsenkirchenstr. 5, 38108, Braunschweig, Germany;

关键词: scFv;    Phage display;    Antibody engineering;    Library;    Panning;    Screening;   

DOI  :  10.1186/s12896-015-0125-0

 received in 2014-11-11, accepted in 2015-02-09,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundAntibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.ResultsThe naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.ConclusionThe highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

【 授权许可】

Unknown   
© Kügler et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.