Generierung und Charakterisierung rekombinanter single chain Fv-Derivate für therapeutische und diagnostische Anwendungen an unterschiedlichen Leukämie Subtypen

Acute myeloid leukemia (AML) is the most common myeloid leukemia with a median age at presentation of about 67 years and an overall survival rate of less than 25%. It is most probably caused by genetic mutations in hematopoietic progenitor cells and maintained by a small population of stem cells. The surface receptor-expression pattern of these leukemic stem cells (LSCs) is well defined and makes these cells attractive targets for diagnosis and immunotherapy. The aim of this PhD thesis was to generate target specific single chain variable fragments (scFv) against three AML-related cell surface receptors (CD33, CD96 and FLT3 (CD135)) via phage display. These isolated scFvs should be analyzed, together with already existing scFvs against CD64, CD30 and CD89, in diagnostic and therapeutic approaches. We could successfully generate three different phage display libraries based on CD33-, CD96- and FLT3 immunized mice. Thereafter, several scFvs specific for each target antigen could be isolated. Subsequently, the scFvs were expressed in E. coli, purified and characterized. Affinity studies via flow cytometry showed equilibrium binding constants in a low nanomolar range. Finally, the most promising scFvs were used to generate recombinant scFv-based fusion proteins, such as scFv-SNAP fusions, Pseudomonas aeruginosa exotoxin A (ETA')-based immunotoxins, human cytolytic fusion proteins (hCFPs) and single chain triple bodies (sctb). After expression in HEK293T-cells these constructs were tested for their biofunctionality where we could successfully show specific binding of our fusion-proteins to the respective target cell-lines. We established an ADCC-assays with isolated peripheral blood mononuclear cell (PBMCs) where we were able to demonstrate specific killing of the target cell lines induced by our CD33 specific sctbs. Additionally, we designed and characterized two CD89-based hCFPs containing the human microtubule-associated protein tau (MapTau) or a mutant form of human angiogenin (AngGGRR). A CD89-based immunotoxin αCD89(scFv)-ETA', containing a truncated version of ETA', induced apoptosis in primary cells from patients with AML. However, immunotoxins containing bacterial or plant toxins suffer from several drawbacks, including immunogenicity. We could show that our novel constructs eliminate primary cells from patients suffering from AML, acute myelomonocytic leukaemia (AMML) and chronic myeloid leukaemia (CML), more efficiently than αCD89(scFv)-ETA'. Both hCFPs successfully induced apoptosis in leukaemic cell lines and primary cells from eight patients suffering from different subtypes of leukaemia. A direct comparison confirmed that our hCFPs perform better than αCD89(scFv)-ETA' and therefore prove suitable for the development of novel immunotherapeutic agents. In addition to the therapeutic part of this thesis, two CD96-specific scFvs recognizing denatured CD96 could be isolated, enabling detection of the target antigen via Western Blot and in fixed blood smear or bone marrow slides via microscopy. Additionally, equilibrium binding constants in low nanomolar ranges could be demonstrated for these fusions. CD33- and FLT3- specific scFv-SNAP fusions were generated and characterized as well. These constructs are opening a broad range of potential applications, e.g. FACS analysis for the diagnosis AML-subtypes. Within the last two decades, nanotechnology applications have gotten a lot of attention in cancer therapy and diagnosis. Silica based nanoparticle (SiNPs) are easy to synthesize, have adjustable pore volume, uniform morphology and a modifiable surface. We received two different ATTO647N fluorescence labeled SiNPs from our cooperation partners (Fraunhofer ISC), hereof one was modified with a BG-Tag or a tert-Butyloxycarbonyl (BOC) protecting group, respectively. First experiments showed a strong unspecific staining of cells caused by the nanoparticles. We could reduce 95% of this effect by blocking the nanoparticles prior to all experiments. Thereafter we were able to show a specific coupling of SNAP-tagged scFvs to the BG-modified nanoparticles, whereas there was no reaction with the BOC-modified nanoparticles. Additionally, a serial dilution was performed, and optimal nanoparticle concentrations titrated. We could show a specific signal in dilutions with up to ~20 ng of protein. Similar results could be obtained with SiNP-fusions containing scFvs against CD30, CD33 and CD96. Specific binding to the target cell lines could be shown for all coupled scFvs, while there was no binding to the negative cell lines. We are convinced these SiNPs, combined with our scFvs against AML related markers, can provide new applications in both cancer diagnostic and therapy