Das tumortherapeutische Potential optimierter Agonisten der Rezeptoren des Apoptose-induzierenden Liganden TRAIL

Trotz der Entwicklung neuer Wirkstoffen und verbesserter Therapien ist die Bekämpfung von Tumoren weiterhin eine große Herausforderung. Apo-2L oder „tumor necrosis factor (TNF)-related apoptosis ligand“ (TRAIL), TRAIL, ist ein Mitglied der TNF-Familie und ein vielversprechendes Protein für die Krebs...

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Bibliographic Details
Main Author: Schader, Manuela
Contributors: Eilers, Martin (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
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Despite the development of new drugs and the improvement of therapies, the fight against cancer is still ongoing. Apo-2L or tumor necrosis factor (TNF)-related apoptosis ligand, TRAIL, is one of several members of the TNF family and due its selectivity towards transformed versus normal cells it bears the potential as a cancer therapeutic agent. Furthermore, the TRAIL system seems to circumvent the resistance of tumors induced by treatment with chemo- and radiotherapie. In this study multivalent single chain antibody fragments (scFv fragments) specific for TRAIL-R2 and CD95 were generated and analysed. CDNA was prepared from isolated RNA of hybridoma cells expressing monoclonal antibody for TRAIL-R2 and CD95 (APO-1/Fas) and used for PCR amplification of heavy and light variable regions (VH and VL) genes. The VH and VL expression cassettes were C-terminally fused to a multimerisation domain of a modified leucine-zipper. The recombinant proteins were produced by transformation of E.coli with the generated expression leucine zipper plasmids. Specificity and activity of the in-vitro-refolded scFv-anti-TRAIL-R2 and scFv-anti-APO-1 antibody fragments have been evaluated. The specific binding of the scFv-anti-TRAIL-R2 fragment to TRAIL-R2 expressing tumor cells was confirmed by FACS analysis. Only minimal lytic activity of the scFv-anti-TRAIL-R2 fragment on a variety of tumor cells could be detected. In the case of the scFv-anti-APO-1 fragments, specific binding as well as lytic activity was observed. However, compared to the parental anti-APO-1 antibody the potential of this recombinant antibody was not increased. In addition, a highly active recombinant version of TRAIL, IZ-TRAIL, was generated in this study. IZ-TRAIL was recombinantly in E.coli expressed and purified in large scale by sequential affinity chromatography. The anti-tumoral efficacy of IZ-TRAIL alone or in combination with different chemotherapeutic drugs was evaluated on a variety of tumor cells. Besides the high apoptosis-inducing activity on cancer cells, no toxicity in mice and on freshly isolated human hepatocytes of either IZ-TRAIL alone or in combination with diverse chemotherapeutic drugs was observed. Therefore, in contrast to non-tagged versions of TRAIL this recombinant IZ-TRAIL shows high activity against tumor cells and, at the same time, it is not toxic to primary cells.