title: Generation and analysis of the improved human HAL9/10 antibody phage display libraries.
abstract: BACKGROUND
  Antibody 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.
  
  
  RESULTS
  The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. jonas.kuegler@tu-bs.de.|mAb-factory GmbH, Gelsenkirchenstr. 5, 38108, Braunschweig, Germany. jonas.kuegler@tu-bs.de.
authors.forename: Jonas
authors.initials: J
authors.lastname: Kügler
authors.affiliation: mAb-factory GmbH, Gelsenkirchenstr. 5, 38108, Braunschweig, Germany. sonja.wilke@mab-factory.com.
authors.forename: Sonja
authors.initials: S
authors.lastname: Wilke
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. d.meier@tu-bs.de.
authors.forename: Doris
authors.initials: D
authors.lastname: Meier
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. f.tomszak@tu-bs.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Tomszak
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. andre.frenzel@tu-bs.de.|YUMAB GmbH, Rebenring 33, 38106, Braunschweig, Germany. andre.frenzel@tu-bs.de.
authors.forename: André
authors.initials: A
authors.lastname: Frenzel
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. th.schirrmann@tu-bs.de.|YUMAB GmbH, Rebenring 33, 38106, Braunschweig, Germany. th.schirrmann@tu-bs.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. s.duebel@tu-bs.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.affiliation: Klinikum Braunschweig g GmbH, Institut für Klinische Transfusionsmedizin, Celler Str. 38, 38114, Braunschweig, Germany. h.garritsen@klinikum-braunschweig.de.|Department Vaccinology, Helmholtz-Zentrum für Infektionsforschung, Inhoffenstraße 7, 38124, Braunschweig, Germany. h.garritsen@klinikum-braunschweig.de.
authors.forename: Henk
authors.initials: H
authors.lastname: Garritsen
authors.affiliation: Merck KGaA, Darmstadt, Germany. Bjoern.Hock@merckgroup.com.
authors.forename: Björn
authors.initials: B
authors.lastname: Hock
authors.affiliation: Merck KGaA, Darmstadt, Germany. lars.toleikis@merckgroup.com.
authors.forename: Lars
authors.initials: L
authors.lastname: Toleikis
authors.affiliation: Merck KGaA, Darmstadt, Germany. Mark.Schuette@merckgroup.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Schütte
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. m.hust@tu-bs.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
mesh_terms: D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D001324:Autoantigens; C063762:CDR1 protein, human; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D057127:Single-Chain Antibodies