Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate

A model compound of anti-tumor agent, segment B of duocarmycin derivative D U-86, was conjugated to tumor-specific antibody via a cleavable linker cons isting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala -Val), to confirm the feasibility of the linker for application to immunoco njugate. The release of segment B from the linker was evaluated by HPLC ana lysis. When segment B was derivatized to have an amino residue and then lin ked to PEG through a dipeptide, segment B was cleaved at the peptide bond b y a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3. 4.2 1.7.), indicating that certain protease specifically expressed at the t umor site would be capable of peptide-specific digestion and release of ant i-tumor agent since a thermolysin-like enzyme has been reported to be expre ssed at many tumor cells. Furthermore, the results showing that cell extrac t from G361 human melanoma had an ability to digest the linker peptide whil e the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form im munoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specifi c antibody and tumor specific activation of the anti-tumor agents to enhanc e tumor treatment efficacy and to decrease unwanted side effects. (C) 2000 Elsevier Science B.V. All rights reserved.