Ton Logtenberg

Exploiting the Natural Diversity in Adenovirus Tropism for Therapy and Prevention of Disease

ABSTRACT Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

High-level expression of recombinant IgG in the human cell line PER.C6

The number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 × 106 cells/mL and produce 300–500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody‐producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1–10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high‐mannose structures are seen.

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

New perspectives on recombinant human antibodies

textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies.

Abstract We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.

Use of phage display antibodies for monitoring cytokine-induced priming of granulocytes in human peripheral blood

Use of phage display antibodies for monitoring cytokine-induced priming of granulocytes in human peripheral blood E. Fortunatia,∗, D. Kantersa, J.W.J. Lammersa, J. de Kruifb,c, T. Logtenbergb,c and L. Koendermana Dept. Pulmonary Disease and Immunology, University Medical Centre, Utrecht, The Netherlands Utrecht Biotechnology Systems, Utrecht, The Netherlands The development of the phage display technique has led to the possibility of in vitro production of human antibodies. The most challenging medical application is their use in the identification of associated disease proteins. Our research interest is focused on the understanding of human granulocyte pre-activation (priming) in vivo. This process is extremely important in the control of host defence against pathogenic microorganisms. However, uncontrolled activation may lead to diseases such as adult respiratory stress syndrome and septic shock [1]. Granulocyte activation can be induced in vitro by the addition of pre-activating (priming) substances like cytokines, chemokines and/or bacterial products. After screening of a semi-synthetic phage antibody library of human scFv fragments, two human phage antibodies named MoPhab A17 and A27, were selected for ∗Correspondence to: Elisabetta Fortunati, Department Pulmonary Diseases, University Medical Centre, Heidelberglaan 100, Post G.03.550, 3584 CX Utrecht, The Netherlands. Fax: +31 3