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 p rolong circulating half-life of proteins. In the present study, site-direct ed PEGylation was used to reduce immunogenicity and prolong plasma half-lif e of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K17 3C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifica tions were done by reacting PEG(5k)-maleimide or PEG(20k)-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clea rance rate of PEGylated TCS mutants decreased up to 100-fold and the decrea se was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were al so decreased. However, the in vivo abortifacient activity was, slightly dec reased, unchanged, or even enhanced in some preparations. PEG(5k) modificat ion had little effect on immunogenicity. However, PEG(20k) modification sig nificantly 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 conc lusion, the present results suggest that PEG(20k) is better than PEG(5k) fo r reducing immunogenicity and prolonging plasma half-life. The conjugate ca n become a better therapeutic agent.