Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity

Bioconjug Chem. Jul-Aug 2002;13(4):855-69. doi: 10.1021/bc025536j.


The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / pharmacokinetics*
  • Antigens, Neoplasm / immunology
  • Cathepsin B / metabolism*
  • Cell Division / drug effects
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / metabolism*
  • Dipeptides / chemistry
  • Dipeptides / metabolism*
  • Doxorubicin / chemical synthesis
  • Doxorubicin / pharmacokinetics*
  • Drug Stability
  • Enzymes / metabolism
  • Humans
  • Kinetics
  • Lewis Blood Group Antigens / immunology
  • Lysosomes / enzymology
  • Lysosomes / metabolism*
  • Rats
  • Tumor Cells, Cultured


  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • BR96-doxorubicin immunoconjugate
  • Cross-Linking Reagents
  • Dipeptides
  • Enzymes
  • Lewis Blood Group Antigens
  • Lewis Y antigen
  • Doxorubicin
  • Cathepsin B