A model compound of anti-
tumor agent, segment B of
duocarmycin derivative
DU-86, was conjugated to
tumor-specific antibody via a cleavable linker consisting of poly(
ethylene glycol) (PEG) and
dipeptide, L-alanyl-
L-valine (Ala-Val), to confirm the feasibility of the linker for application to
immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a
dipeptide, segment B was cleaved at the
peptide bond by 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
tumor site would be capable of
peptide-specific digestion and release of anti-
tumor agent since a
thermolysin-like
enzyme has been reported to be expressed at many
tumor cells. Furthermore, the results showing that
cell extract from G361 human
melanoma had an ability to digest the linker
peptide while 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
immunoconjugate 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 specific antibody and
tumor specific activation of the anti-
tumor agents to enhance
tumor treatment efficacy and to decrease unwanted side effects.