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

Abstract

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.

The invasin D protein from Yersinia pseudotuberculosis selectively binds the Fab region of host antibodies and affects colonization of the intestine

“…Characterization of Fab binding specificity-To gain a detailed insight into the interaction of InvD with Fab on the molecular level, we performed phage display panning. Two scFv naive universal libraries (HAL10 and HAL9+HAL10) were employed (32). Three rounds of panning were performed on immobilized strep-tagged InvD1640 followed by enzyme-linked immunosorbent assay (ELISA) with soluble single chain fragment variable (scFv).…”

“…scFv phage library panning-HAL9 and HAL10 scFv libraries with a size of 1.04x10 10 and 4.45x10 9 respectively were used for panning (32). Panning and production of soluble scFv was performed according to (89).…”

Designing Human Antibodies by Phage Display

“…These 'universal' or 'single-pot' libraries must contain a repertoire of antibody genes several orders of magnitude more diverse compared to immune libraries in order to maintain good chances to provide appropriate binders for all possible antigen structures. Successful universal libraries typically contain more than 10 billion different antibody clones [32], making today's antibody phage display libraries the world's largest gene collections by a huge margin.…”

Human Anti-Lipopolysaccharid (LPS) antibodies against Legionella with high species specificity

“…Phage (∼1 × 10 11 cfu) of the HAL9/10 libraries [30] were incubated with the formaldehyde fixed P. aeruginosa cells (∼1 × 10 9 cells) for 1 h at room temperature in suspension. Cell bound phage were removed by centrifugation (10,000 g, 5 min), the supernatant was collected and cleared from the remaining cells by filtration (0.2 µm).…”

“…In this work, recombinant monoclonal antibodies were selected from the naive human phage display libraries HAL9/10 [30] by panning on whole L. pneumophila cells. Sandwich pairs were designed and used in portable immunosensor for the detection of Legionella.…”