Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates

J Med Chem. 2005 Mar 10;48(5):1344-58. doi: 10.1021/jm040137q.


Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antineoplastic Agents, Alkylating / chemical synthesis*
  • Antineoplastic Agents, Alkylating / chemistry
  • Antineoplastic Agents, Alkylating / pharmacology
  • Aza Compounds / chemical synthesis
  • Aza Compounds / chemistry
  • Aza Compounds / pharmacology
  • Cathepsin B / chemistry
  • Cell Line, Tumor
  • Cyclopropanes / chemical synthesis
  • Cyclopropanes / chemistry
  • Cyclopropanes / pharmacology
  • DNA / chemistry*
  • Dipeptides / chemistry*
  • Drug Design
  • Drug Screening Assays, Antitumor
  • Humans
  • Immunoconjugates / chemistry*
  • Indoles / chemical synthesis*
  • Indoles / chemistry
  • Indoles / pharmacology
  • Mice
  • Structure-Activity Relationship


  • 1,2,9,9a-tetrahydrocyclopropa(c)benz(e)indol-4-one
  • Antibodies, Monoclonal
  • Antineoplastic Agents, Alkylating
  • Aza Compounds
  • Cyclopropanes
  • Dipeptides
  • Immunoconjugates
  • Indoles
  • DNA
  • Cathepsin B