Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation.

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 microg/mL while no effect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe(a) expression.