Konstruktion und Charakterisierung von rekombinanten Immuntoxinen zur Therapie von metastasierendem Pankreaskarzinom

Pancreatic carcinoma is the fifth leading cause of cancer-related deaths in North America and Europe. Major reasons for the high mortality rate are the inability to detect pancreatic cancer at an early stage, extensive local invasion and early formation of lymphatic and hematogenous metastases. Including the conventional treatments for cancer like surgery, chemotherapy and radiotherapy, there is presently no efficient therapy for advanced pancreatic carcinoma which might be able to significantly improve the outcome of the patients. Main problems are multiple unacceptable side effects resulting from high dose chemotherapy and a strong resistance of pancreatic carcinoma to both, chemo- and radiotherapy. Consequently, novel effective therapies have to be urgently developed. Immunotherapy, comprising the application of monoclonal antibodies, immunotoxins or bispecific antibodies, is a promising alternative for treatment of pancreatic carcinoma. Since overexpression of EGF receptor (EGFR) in pancreatic tumors correlates with advanced clinical staging, tumor size and decreased survival of patients, it represents an appropriate target for antibody-based immunotherapy. In this thesis the construction and characterisation of several EGFR-specific recombinant immunotoxins with regard to a possible therapy of pancreatic carcinoma is described. First of all the recombinant immunotoxin 425(scFv)-ETA’ was generated by genetically fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas exotoxin A (ETA’). Using the expression vector pBM1.1, functional 425(scFv)-ETA’ was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes. Binding activity of the purified immunotoxin to the EGFR-positive pancreatic carcinoma cell lines L3.6pl and FG was confirmed by flow cytometry, ELISA and immunofluorescence-microscopy. XTT-based cell proliferation assays showed that the toxic domain of the fusionprotein was functional as well. The median IC50, which is the concentration of a protein leading to 50% inhibition of cell proliferation, was calculated to be 0,0075 µg/ml for L3.6pl cells and 0,008 µg/ml for FG cells, respectively. Furthermore the anti-tumor effect of this recombinant immunotoxin was confirmed using a disseminated human pancreatic cancer nude mouse model. After intravenous injection of L3.6pl cells into immunodeficient nude mice, both, single and repeated intravenous administration of 425(scFv)-ETA’ resulted in a significant reduction of the average amount of lung metastases, verifying the anti-tumor activity of 425(scFv)-ETA’ in vivo. To reduce the immunogenicity of anti-EGFR immunotoxins, fusionproteins containing human enzymes (hE) as toxic domains were generated in the second part of this doctoral thesis. Therefore 425(scFv) was genetically fused to the N- and C-termini of recombinant angiogenin (Ang), eosinophilic cationic protein (ECP) and granzyme B (Gb) by insertion into the eucaryotic expression vector pMS. A leader sequence was mediating the secretion of the 425(scFv)-hE and hE-425(scFv) immunotoxins into the cell culture supernatant of eucaryotic HEK293T cells. After purification by metal-ion affinity chromatography the binding activity of the fusionproteins to the EGFR-positive cell lines L3.6pl and FG was confirmed by flow cytometry and ELISA. Cytotoxicity assays showed that 425(scFv)-Ang and 425(scFv)-Gb exhibited specific cytotoxicity against L3.6pl cells reaching an IC50 of 0,380 µg/ml and 0,430 µg/ml, respectively. In contrast, the corresponding immunotoxins with N-terminal located hE, Ang-425(scFv) and Gb-425(scFv), showed a lower cytotoxic activity towards pancreatic carcinoma cells. A possible reason for the lower IC50 of 425(scFv)-Ang and 425(scFv)-Gb compared to the IC50 of 425(scFv)-ETA’ could be an inadequate translocation of the effector domains into the cytosol, as in contrast to ETA’ the human enzymes naturally contain no translocation domain. Accordingly anti-EGFR specific immunotoxins were generated, containing a truncated mutant of the translocation domain of ETA’ at the N-terminus of hE. The resulting fusionproteins 425(scFv)-dII-Ang and 425(scFv)-dII-Gb were successfully expressed in HEK293T cells and possessed binding activity towards the EGFR. However, the cytotoxicity of 425(scFv)-Ang and 425(scFv)-Gb could not be improved by insertion of the translocation domain. In summary, this thesis shows that the generated recombinant anti-EGFR immunotoxins are promising candidates for the treatment of metastatic pancreatic carcinoma. In particular, 425(scFv)-ETA’ exhibited in vivo a strong anti-tumor effect and might be suitable for further clinical investigation