Towards the generation of recombinant and pharmaceutically relevant target proteins : phage display based isolation of specific scFv antibodies against metastasizing pancreatic carcinoma for clinical application and development of novel fluorescent reporter tags for on-line monitoring during target protein production

• Über die Generierung rekombinanter und pharmazeutisch relevanter Zielproteine : die Phage-Display-basierte Isolierung spezifischer scFv-Antikörper zur klinischen Anwendung gegen das metastasierende Pankreaskarzinom und die Entwicklung eines neuartigen fluoreszierenden Reportertags zum Online-Monitoring der Produktion eines Zielproteins

Siepert, Eva-Maria; Barth, Stefan (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2013, 2014)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2013

Abstract

Pancreatic cancer is an aggressive type of neoplasia characterized by its high potential for metastasis with a most devastating prognosis. Initial stages are almost asymptomatic, thus preventing early detection before local tissue invasion due to the lack of reliable diagnostics. Surgical removal in combination with standard first-line chemotherapeutic Gemcitabine treatment and radiation-based therapy are merely life-prolonging options. High resistance towards conventional therapeutics and the huge metastasizing potential leaves minimal residual micrometastasis accountable for an enormously high relapse rate. Therefore, the first aim of this thesis was to develop novel tumor specific human single chain antibody fragments (scFv) for possible future application as cytolytic therapeutics for adjuvant treatment of metastasizing pancreatic cancer, as well as more efficient molecular tools for early diagnosis. Phage display technology was employed to generate pancreas-specific scFv-phage antibodies from the naïve human Tomlinson phage libraries I and J binding against unknown tumor-associated antigen. Highly specific scFv phage ligands were isolated in a two-step panning strategy via depletion on human peripheral blood mononuclear cells (PBMC), followed by a positive selection on the metastatic pancreatic cancer cell line L3.6pl. Monoclonal phage ELISA identified 16 unique L3.6pl positive scFvs, subsequently expressed in eukaryotic HEK293T cells as soluble scFv-SNAP-tag proteins. Additionally, clone 14.1(scFv) SNAP, originally isolated from a laboratory-own murine immunized phage display library, was included into the expression and characterization process. Analysis of binding specificity and cross-reactivity of nine IMAC purified scFv-SNAP proteins was performed by soluble protein ELISA and flow. Of these, four clones displayed internalizing properties during flow cytometric and OPERA-based internalization tests. All positive candidates are clinically relevant pancreatic carcinoma specific scFvs and may provide the prospect of a tumor targeted cancer therapy to eliminate residual cancer cells. Moreover, they are highly promising candidates for diagnostic in vivo imaging tools besides an additional application as theranostics. To produce recombinant pharmaceutically relevant proteins on large scale, highly efficient screening technologies have been developed to characterize optimum cultivation conditions for bacterial growth and production formation. Microtiter plates (MTPs), in combination with measurement systems such as the BioLector®, are a practical tool for non invasive on-line monitoring of product formation of continuously shaken microbial cultures on lab-scale. Conventional reporter proteins for on-line monitoring, such as GFP and its derivatives, or flavin mononucleotide-based fluorescent proteins, are very large which possibly imposes stress on the host organism. Additionally, GFP strongly depends on an oxygen saturated environment for fluorophore formation. To circumvent mentioned drawbacks of conventional reporter tags, short but still optically active reporter tag for on-line detection were designed in this thesis. These novel reporter tags (W-tags) are based on the auto fluorescence of the aromatic amino acid tryptophan. Using in silico techniques, between one and five tryptophan residues (W1-W5) were accumulated in the naturally occuring protein loop of the cold shock protein (Bc Csp), to have equilibrated charges with the tryptophan residues presented on the outer side of the loop. Genetic fusions of these five different W-tags (MW = 3.4 to 5.6 kDa) to the anti-CD30 Ki-4(scFv) as well as the anti-MucI M12(scFv) antibody fragment were produced in a prokaryotic pMT-expression system. Analysis of on-line product fluorescence intensity during fermentation in MTPs followed by molecular biological flow cytometric binding analysis showed that more tryptophan residues within a W-tag generated a stronger tryptophan fluorescence signal gaining intensity corresponding to product formation. Nevertheless, an increase in tryptophan residues also complicated concentration of W-tagged proteins in the cell lysate. Protein recovery was only possible for constructs W1-W3. W4 and W5 remained in the cell pellet due to highly hydrophobic properties of the accumulated tryptophan molecules. Normal and comparative flow cytometry of W-tagged Ki-4(scFv) proteins on L540cy cells, in combination with a Ki-4 full length antibody, confirmed that binding specificity was not influenced whereas W tags with more than one tryptophan residue seemed to have a negative effect on binding activity and affinity. Lacking the main drawbacks of conventional reporter proteins, the novel W tags are an applicable alternative during non-invasive monitoring of recombinant product formation. They present the possibility for rapid and qualitative on line measurement during large-scale production of pharmaceutically relevant target proteins.

Institutions

• [811001-1]