Development an engineered human enzyme for anti-vasculature immunotherapy

Abstract

The tumour vasculature is an attractive target for cancer therapy because tumour growth and spread depend on these structures for oxygen and nutrient supply. Anti-vasculature therapy aims to cause rapid and selective shutdown of the established tumour vasculature, leading to secondary tumour cell death. In this study, we propose to employ an Antibody Guided Enzyme Nitrile Therapy (AGENT) approach to selectively target extra-domain B fibronectin (B-FN), which is abundantly expressed in tumour vasculature. A recombinant fusion protein, composed of the humanised BC-1 single chain fragment (huBC1 scFv) fused with a re-engineered human cytosolic β–glucosidase (rehCβG) enzyme, was investigated to target this antigen and generate cytotoxic cyanide molecules specifically within tumour sites. Naturally-occurring human β-glucosidase (hCβG) cannot hydrolyse the cyanogenic glycoside linamarin, to any significant level, but was chosen as a suitable candidate for site-directed mutagenesis due to its homology with a native cyanogenic plant enzyme, linamarase. Both huBC-1 scFv and hCβG protein were expressed in Escherichia coli and purified by affinity and size exclusion chromatography. In vitro characterisation of the huBC-1 scFv showed that it retained antigen-binding specificity of the parental antibody, Immunoglobulin-G HuBC-1 towards the same antigen. Kinetic analysis of the wild type and a panel of mutant hCβGs showed that their catalytic activity was retained. Interestingly, one of the mutants, V168A hCβG, was shown to have increased cyanogenic activity towards linamarin. The Km was improved from 2.23 ± 0.03 mM to 0.79 ± 0.02 mM and the kcat from 0.21 ± 0.01 min-1 to 5.49 ± 0.01 min-1. Following this discovery, construction and expression of the fusion protein, scFv-rehCβG (wild-type and V168A) was carried out using different expression systems to compare the best strategy in constructing the fusion protein. The huBC-1 scFv-hCβG fusion proteins were expressed cytosolically but as insoluble aggregates in inclusion bodies. Recovery of the fusion protein was made through refolding of the protein. Using the dilution method, the protein was solubilised and refolded, however it was functionally inactive. Another refolding method via dialysis experienced loss of the fusion protein. Final production of a cyanogenic scFv-hCβG requires further investigation by optimising the protein refolding protocol which could lead to further studies of AGENT.