Charakterisierung und Evaluierung neuartiger Antikörperfragment-Immunkonjugate basierend auf der SNAP - Tag Technologie

Treatment of cancer, especially of solid tumors, is challenging and today classical chemotherapy is often supported by antibody-based therapies. This includes antibody-drug-conjugates (ADCs), which combine the specificity of monoclonal antibodies and high toxic impact of effector molecules, like microtubule disrupting agents. Two of them have been approved for cancer treatment. Until today, ADCs are generated by random conjugation methods that do not allow a defined stoichiometry. Therefore new site-directed conjugation strategies are of high interest for the generation of novel ADCs. Within the scope of this thesis novel antibody fragment-based immunoconjugates were generated using the SNAP-tag technology. This technology permits a site-directed conjugation of benzylguanine (BG) modified effector molecules to antibody fragments, allowing the synthesis of a homogenous and defined conjugate. The high molecular weight of 150 kDa of full-length antibodies reduce the tumor penetration of ADCs especially in solid tumors. Therefore antibody fragments, like single-chain Fragments variable (scFvs), were used as antigen-binding moiety of the novel ADCs, to increase tumor penetration and pharmacokinetics. Several of solid tumors such as mamma carcinoma, showed overexpression of members of the epidermal growth factor receptors family, specifically EGFR (epidermal growth factor receptor 1) and HER2 (human epidermal growth factor receptor 2). Thus, novel ADCs based on EGFR- and HER2-specific scFv-SNAP fusion proteins were generated and characterized. As an ADC subform the before established photo-immunoconjugate, consisting of the EGFR-specific 425(scFv)-SNAP fusion protein and the photosensitizer Chlorin e6 (Ce6), was evaluated regarding its in vivo functionality in a murine model. Therefore the BG modification of Ce6 was successfully optimized to generate a sufficient amount of photo immunoconjugate. In following experiments the in vivo functionality of the conjugate, regarding its best suitable irradiation time point, the optimal irradiation dose and the optimal dose of photo immunoconjugate, was investigated. Although 425(scFv)-SNAP-Ce6 showed an EC50-value of 58 nM in vitro when irradiated with 25 J/cm², no tumor regression could be observed and determined in vivo due to suboptimal tumor growth. Therefore the experiment was aborted in respect of animal welfare. Furthermore novel SNAP-tag based ADCs were generated, conjugated to microtubule disrupting agents. Three different microtubule disrupting agents, tubulysin, nocodazole and auristatin F, were conjugated to the EGFR-specific and/or HER2-specific scFv-SNAP fusion proteins and cytotoxic potential of the novel ADCs was evaluated in vitro. In line with this work eight different tubulysin constructs, which differ in length und cleavability of their linker structure, were also evaluated. The results demonstrated that the functionality of the novel tubulysin-based ADCs depends on the linker structure. The conjugation of tubulysin derivatives with short linker structures to scFv-SNAP fusion proteins resulted in unspecific toxicity, independently if the linker structure was non-cleavable or cleavable. Only the conjugation of one tubulysin derivative, TAM816d, which was modified with a PEG10 linker, showed specific cytotoxicity and induced apoptosis in the lower nanomolar range when conjugated to both scFv-SNAP fusion proteins. The positive influence of a longer PEG-based linker (PEG24) could be verified by performing experiments with another microtubule inhibitor named nocodazole. After conjugation of nocodazole to 425(scFv)-SNAP the directed elimination of target cells could be demonstrated. The generation of two additional potent ADCs, consisting of auristatin F (AURIF) modified with a PEG1 linker and an EGFR- or HER2-specific scFv-SNAP fusion, demonstrated significant cytotoxic and pro-apoptotic effects as well as induction of cell cycle arrest on selected s mamma carcinoma cell lines. The resulting EC50 values verified, that the toxicity of the ADCs in comparison to the free toxin was not impaired or even increased after conjugation of the toxin to the fusion proteins. For 425(scFv)-SNAP-AURIF an EC50 value of 3 nM and for αHER2(scFv)-SNAP-AURIF of 0.6 nM could be determined. Besides high serum stability up to 48 h, which is important for potentially in vivo applications, specific ex vivo binding of 425(scFv)-SNAP-AURIF to EGFR-positive breast cancer tumor biopsies could be demonstrated. With these results the promising potential of the novel employed ADCs could be pointed out

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb (TM) technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells

A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR

Purpose: The term “theranostics” represents a new paradigm in medicine especially for cancer treatment. This term was coined by Funkhouser in 2002 and defines a reagent that combines therapeutic and diagnostic properties. It is widely believed that theranostics agents will have considerable impact on healthcare before, during, and after disease by improving cancer prognosis and management simultaneously. Current theranostics approaches still rely on passive tumor targeting strategies, which have scattergun effects and tend to damage both neoplastic and non-neoplastic cells. Methods: Here we describe a simple, controlled, and efficient method to generate homogeneous photoimmunotheranostics reagents. This method combines molecular optical imaging, photodynamic therapy, and immunotherapy using SNAP-tag technology. SNAP-tag is a derivative of the O(6)-alkylguanine-DNA alkyltransferase (AGT) which has the ability to efficiently conjugate to O(6)-benzylguanine (BG) molecules under physiological conditions depending on its folding pattern. Results: The theranostics agent was able to specifically recognize various epidermal growth factor receptor (EGFR)-expressing skin cancer cell lines using flow cytometry analysis and confocal microscopy and eliminate them at EC50’s of 32–55 nM. Conclusions: These experiments provide a framework for using SNAP-tag technology to generate homogeneous photoimmunotheranostics reagents with unified pharmacokinetic and therapeutic profiles. Furthermore, the reagent generated in this work could be used to simultaneously monitor and suppress the growth of skin squamous carcinoma and melanoma cells expressing EGFR.National Research Foundation (South Africa

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes.

In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research

Oligonucleotides for PCR amplification.

<p>Oligonucleotides for PCR amplification.</p

XTT cell viability assay to determine the cytotoxicity of TTC-based proteins.

<p>TTC-reactive REH cells (black) as well as the mock-transfected control REH cells (gray) were used to demonstrate the cytotoxicity of TTC-ETA' (<b>A</b>, ■), GrB(R201K)-TTC (<b>B</b>, ▼) and TTC-MAPTau (<b>C</b>, ●). The cells were incubated with an increasing concentration of the recombinant fusion proteins for 72 h at 37°C and 5% CO₂ followed by an XTT cell viability assay. The EC₅₀ value relative to untreated control cells (100% cell viability) and the positive control zeocin (0% cell viability) was calculated using the three-parameter dose-response curve fit equation with GraphPad Prism v5 software. The data are means ± SD of three independent experiments performed in triplicate (n = 3). Statistical significance was calculated by two-way ANOVA followed by Bonferroni’s post-hoc test (*** p < 0.001, ** p < 0.01, * p < 0.05, n.s.–not significant).</p

Internalization of SNAP-TTC-BG-647 by TTC-reactive REH cells.

<p>The TTC-reactive REH cells were incubated with SNAP-TTC-BG-647 at 4°C (<b>A</b>) or 37°C (<b>B</b>) and the cells were observed by confocal microscopy. As negative controls, TTC-reactive REH cells were incubated without protein (<b>C</b>) and mock-transfected REH cells were incubated with SNAP-TTC-BG-647 for 30 min at 37°C (<b>D</b>). Images of the eGFP (excitation = 488 nm), SNAP-TTC-BG-647 (excitation = 647 nm) and nuclear counterstaining (excitation = 405 nm) signals were merged using ImageJ v1.50f software. Scale bar = 10 μm.</p