Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed PEGylation

PEG modification (PEGylation) has been shown to reduce immunogenicity and prolong circulating half-life of proteins. In the present study, site-directed PEGylation was used to reduce immunogenicity and prolong plasma half-life of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K173C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifications were done by reacting PEG(5k)-maleimide or PEG(20k)-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clearance rate of PEGylated TCS mutants decreased up to 100-fold and the decrease was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were also decreased. However, the in vivo abortifacient activity was, slightly decreased, unchanged, or even enhanced in some preparations. PEG(5k) modification had little effect on immunogenicity. However, PEG(20k) modification significantly reduced immunogenicity. All PEG20k modified TCS mutants induced lower level IgG and IgE antibodies. In particular, PEG(20k)-KQ and PEG(20k)-K173C induced weaker systemic anaphylaxis reaction in guinea pigs. In conclusion, the present results suggest that PEG(20k) is better than PEG(5k) for reducing immunogenicity and prolonging plasma half-life. The conjugate can become a better therapeutic agent.