Engineering Azide Linkers into Fn3 Protein-Drug Conjugates for Targeted Cancer Therapy

Traditional cancer therapies have significant side effects due to the indiscriminate killing of healthy cells alongside cancer cells during treatment. To reduce these side effects, targeted therapies have been developed which are able to distinguish cancer cells from healthy cells based on specific biomarkers. Protein-drug conjugates (PDCs) are a common form of targeted therapy, consisting of a cancer-targeting protein, a cancer-eliminating payload, and a chemical linker to connect protein to payload. PDCs in the Moore Lab use fibronectin type III (Fn3) non-antibody protein scaffolds which are small, easily modifiable, and cost-effective to produce in bacteria. Previously, Fn3 proteins were engineered to target cancer cells by binding to ɑvβ3 integrin receptors with high affinity via an RGD amino acid sequence; these are referred to as FnRGD proteins. ɑvβ3 integrins are overexpressed in tumor cells and tumor neovasculature making them a good biomarker for cancer, such as for glioblastoma, and a therapeutic target. In this current work, a linker is developed to conjugate FnRGD to a payload. This thesis aims to incorporate an azide linker at the C-terminus of the FnRGD protein, enabling efficient, site-specific, and bioorthogonal conjugation of payloads—such as a chemotherapeutic drug, a polymer-drug complex, or a fluorophore—containing an alkyne via click chemistry. In this thesis, two methods for azide linker incorporation were successfully developed and the azide-containing Fn3 proteins were characterized. The first approach incorporated azidohomoalanine (AHA), a non-canonical amino acid with an azide in its side chain, into FnRGD to produce FnRGD-AHA. AHA is translationally incorporated using methionine-auxotrophic bacteria which can replace methionine (Met) with AHA during protein synthesis due to Met being the canonical analog of AHA. Plasmids encoding for FnRGD-AHA were generated using recombinant DNA cloning and expressed using B834(DE3) bacteria. AHA incorporation was verified through mass spectrometry. In the second approach to add an azide, a FnRGD variant with a cysteine at the C-terminus (FnRGD-Cys) was reacted with a maleimide-PEG3-azide compound using thiol-maleimide chemistry. FnRGD-Cys was produced and maleimide-PEG3-azide was synthesized and successfully conjugated to FnRGD-Cys. The presence and functionality of the azide on the proteins were verified through direct labeling with a DBCO-containing fluorophore and by analyzing the labeled proteins using fluorescent protein gels. The azide-containing proteins maintained ɑvβ3 integrin binding ability as verified through flow cytometry with ɑvβ3 integrin-expressing cells. Overall, incorporating an azide linker into the FnRGD protein contributes to creating a novel PDC platform that combines the advantages of small, cost-effective Fn3 proteins with bioorthogonal, efficient azide-alkyne click chemistry.