Florian Tomszak

Bio: Florian Tomszak is an academic researcher from Braunschweig University of Technology. The author has contributed to research in topics: Phage display & Phagemid. The author has an hindex of 4, co-authored 4 publications receiving 147 citations.

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

Abstract: 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. The 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. 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.

Construction of Human Immune and Naive scFv Libraries

Abstract: Antibody phage display is the most commonly used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The prerequisite for successful generation of antibodies using phage display is the construction of high-quality antibody gene libraries. Here, we give the detailed methods for the construction of human immune and naive scFv gene libraries.

Selection of Recombinant Human Antibodies.

Abstract: Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

Designing Human Antibodies by Phage Display

Abstract: With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategies, including the adjustment of kinetic parameters.

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies

Abstract: The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Phage display-derived human antibodies in clinical development and therapy

Abstract: Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity.Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, ada...

Opportunities for therapeutic antibodies directed at G-protein-coupled receptors

Abstract: G-protein-coupled receptors (GPCRs) are activated by a diverse range of ligands, from large proteins and proteases to small peptides, metabolites, neurotransmitters and ions. They are expressed on all cells in the body and have key roles in physiology and homeostasis. As such, GPCRs are one of the most important target classes for therapeutic drug discovery. The development of drugs targeting GPCRs has therapeutic value across a wide range of diseases, including cancer, immune and inflammatory disorders as well as neurological and metabolic diseases. The progress made by targeting GPCRs with antibody-based therapeutics, as well as technical hurdles to overcome, are presented and discussed in this Review. Antibody therapeutics targeting C-C chemokine receptor type 4 (CCR4), CCR5 and calcitonin gene-related peptide (CGRP) are used as illustrative clinical case studies.