Generierung, Charakterisierung und Funktionalisierung rekombinanter Antikörperfragmente gegen leukämische und dendritische Zellen : Entwicklung diagnostischer und immuntherapeutischer Werkzeuge mit Hilfe der „Phage Display“-Technologie

Over the last years, cancer immunotherapy has gained great significance in adjuvant settings and first-line treatment of oncologic malignancies. Therein, monoclonal antibodies, their fragments and conjugates play a crucial part in specific immunotherapy and have now become a part of standard cancer treatment. Due to their ability to specifically identify their target antigen, they reliably differentiate between benign and malignant cells. Hematologic malignancies as the acute myeloid leukemia (AML) for example, are ideal for a treatment with antibody-based drugs. The reason is that malignant cells are often intravascular and therefore accessible to intravenously administered immunotherapeutic agents. Although the cancer treatment regime has been improved significantly over the last decades, the 5-year survival rate is still only 24%. The high mortality correlates with a high relapse rate after complete remission leading to a bad prognosis. The major cause for relapse is the minimal residual disease, a small number of leukemic cells that remain after remission. The development of novel AML-specific immunotherapeutic agents would have benefits for the patient´s quality of life and may prolong the relapse-free survival. Therefore, we aimed to generate specific scFv-antibody fragments using the Phage Display Technology. Using a whole cell panning strategy on an AML-derived cell line, we set the selection pressure on internalizing scFv-fragments. Nine sequence unique scFv-antibodies binding to the cell surface were identified, revealing a nanomolar binding affinity as scFv SNAP-tag fusion proteins. Four of them showed a positive staining on formalin-fixed paraffin-embedded (FFPE) bone marrow tissue of AML diagnosed patients in immunohistochemistry. These observations are the first promising results for the development of a novel diagnostic tool in AML. However, the identity of the bound antigen remains unknown and has to be elucidated to assess the relevance of scFv for diagnostic purposes. Moreover, four scFv-antibodies proofed to internalize into the cell cytosol by confocal microscopy and flow cytometry. Consequently, they were fused to a truncated version of Pseudomonas Exotoxin A (ETA) for recombinant immunotoxin construction. The immunotoxin C3(scFv)-ETA was shown to inhibit cell proliferation and to induce apoptosis in AML-derived cells with an IC50 of 265 nM in vitro. Subsequent studies aim to increase the cytotoxic potential of this molecule via either the addition of adjuvant reagents or the upregulation of surface antigen expression. The data obtained in this thesis demonstrate a putative novel immunotherapeutic approach for effective and selective targeting of leukemic cells in AML therapy. In contrast to this passive approach, active cancer immunotherapy induces a patient’s tumor specific immune response. Most clinical and preclinical studies focus on cancer vaccination with systemically administrated dendritic cells (DC), due to their high immunostimulating potential. A major prerequisite for a successful tumor vaccination is an efficient DC migration to the peripheral lymphoid organs. Monitoring the DC migration behavior after their reinjection would give precious insights which might help to improve the vaccination strategy. Therefore, we aimed to select specific scFv-antibodies against canine dendritic cells (cDC) using the Phage Display Technology. After the biopanning on functional membrane fragments of freshly generated cDC (Prof. Junghanß, University of Rostock), we identified eight unique cDC-specific scFv-antibodies in monoclonal phage-ELISA. Two binders designated C2 and C3 confirmed reproducible binding activity in scFv-ELISA when expressed as soluble proteins. The selection was carried out on cDC membrane fragments of only one dog, but future applications will require the cross-reactive binding of several cDC of the same breed. Therefore, we checked for cross-reactivity to allogeneic cell membranes. At least 70% of the tested membranes were specifically bound by the fusion proteins C2(scFv)-SNAP and C3(scFv)-SNAP, thereby demonstrating a positive cross reactivity. Additionally, we checked for internalization behavior in confocal microscopy and observed cellular uptake of C2(scFv)-SNAP when incubated with viable cDC under physiologic conditions. These characteristics would allow a targeted delivery and processing of tumor specific antigen by the DC when fused to the selected C2(scFv). Further studies could evaluate the use of such a recombinant fusion protein for the development of a putative in vivo vaccination strategy