Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension

2018-08-06Fibroblast activation protein (FAP) is a cell-surface protease highly expressed in cancer-associated fibroblasts (CAFs)—a type of cells in the tumor microenvironment that promote the progression of human epithelial cancers. To deplete FAP-expressing CAFs for treating cancer, a recombinant immunotoxin, αFAP-PE38, was recently developed by fusing a single-chain variable fragment (scFv) against FAP with a truncated form of Pseudomonas exotoxin A (PE38). However, the short plasma half-life of αFAP-PE38 has limited its therapeutic efficacy. The aim of this thesis is to modify αFAP-PE38 with polyethylene glycol (PEG), a polymer that has previously been used to extend the plasma half-life of other therapeutic proteins. We have generated PEGylated αFAP-PE38 using two strategies: (1) PEGylation at a cysteine residue, and (2) PEGylation at an unnatural amino acid residue. We find that a PEGylated αFAP-PE38 protein, in which a 10 kDa PEG is attached to a ketone-containing unnatural amino acid in the linker, can specifically bind to human FAP-expressing 293T cells and inhibit their viability in a dose-dependent manner. However, the αFAP-PE38 proteins modified with larger PEG, including the 20 and 30 kDa variants, have lost binding affinity and cytotoxic effect to human FAP-expressing 293T cells, likely due to increased steric hindrance. Further studies are needed for in vivo evaluation of the pharmacokinetic properties and pharmacologic effects of the 10 kDa PEGylated αFAP-PE38 protein