Institution: | 1 MorphoSys AG, Lena-Christ-Strasse 48, 82152 Martinsried, Germany 2 Sandoz Biopharmaceuticals, Hexal Biotech Forschungs GmbH, Keltenring 1+3, 82041 Oberhaching, Germany 3 Department of Pediatric Oncology, Hematology and Immunology, University of Düsseldorf, Germany 4 Institute of Tropical Medicine and Parasitology, Leopoldstrasse 5, 80802 Munich, Germany 5 Institute of Biochemistry, Medical Faculty, Christian Albrecht University Kiel, Otto-Hahn-Platz 9, 24118 Kiel, Germany 6 AbD Serotec, A Division of MorphoSys, Lena-Christ-Strasse 48, 82152 Martinsried, Germany 7 Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland |
Abstract: | This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications. |