Project Details
Projekt
Twofold bio-orthogonal derivatization of antibody fragments by two different C(alpha)-formylglycine generating enzymes for generation of antibody-drug conjugates
Applicants
Professor Dr. Thomas Dierks (†); Professor Dr. Kristian Müller; Professor Dr. Norbert Sewald
Subject Area
Biological and Biomimetic Chemistry
Term
from 2015 to 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 283316721
Tumor therapy is an important research objective and continues to drive the development of novel biopharmaceuticals. Despite a remarkable improvement of our knowledge in cancer biology and the successful approval of several novel targeted therapies, long term disease control or cure remain the exception rather than the rule in oncology. Moreover, currently used anti-tumor drugs are highly cytotoxic and therapies often are associated with severe side effects. Pharmaceutical agents that specifically recognize molecular structures present on tumor cells would be advantageous to target the delivery of a drug to a tumor cell. Recently, there has been an exponential growth in the field of antibody-drug conjugates (ADCs), where a monoclonal antibody (mAb) is linked to a highly cytotoxic drug. A major challenge in ADC production is to reach a high homogeneity with a uniform drug-antibody ratio (DAR) and a defined location of conjugation site. Tumor-targeting antibodies with relative specific cell toxicity are emerging as first line treatments for some tumors, for example Epidermal Growth Factor Receptor (EGFR) overexpressing tumors. The joint project combines three groups from neighboring departments at Bielefeld University with expertise in biochemistry (Dierks: formylglycine-generating enzymes), biotechnology (Müller: enzyme optimization by cell display, antibody engineering), and bioorganic/bioconjugate chemistry (Sewald: peptide synthesis, drug conjugates) in order to develop and optimize a robust enzymatic system for the twofold bio-orthogonal derivatization of model peptides and antibodies applying two different C(alpha)-formylglycine generating enzymes. Antibodies directed against EGFR will be co-expressed with the human C(alpha)-formylglycine generating enzyme FGE in CHO cells. Enzyme activity will be improved by rational design as well as by directed evolution (using cell display selection technology). After ligation of a first moiety (small-molecule drug or fluorophore) by addressing the newly generated formyl group, the antibody will be further converted in vitro by AtsB, a prokaryotic C(alpha)-formylglycine generating enzyme targeting a different sequence tag. The second formyl group will be used for bio-orthogonal introduction of a second, different moiety (a PEG residue or another type of drug). This approach will lead to antibody conjugates with two drugs having different modes of action or other functionalities, e.g. for uptake and distribution studies. Obtained ADCs will be tested in cytotoxicity assays using cells with different EGFR expression levels.
DFG Programme
Priority Programmes
