Cancer Immunology, Immunotherapy

, Volume 38, Issue 2, pp 92–98

Internalization of an intact doxorubicin immunoconjugate

  • Lisa B. Shih
  • David M. Goldenberg
  • Hong Xuan
  • Helen W. -Z. Lu
  • M. Jules Mattes
  • Thomas C. Hall
Original Articles Doxorubicin Immunoconjugate, Carcinoembryonic Antigen Conjugate, Internalization

DOI: 10.1007/BF01526203

Cite this article as:
Shih, L.B., Goldenberg, D.M., Xuan, H. et al. Cancer Immunol Immunother (1994) 38: 92. doi:10.1007/BF01526203

Abstract

An immunoconjugate between doxorubicin and anti-(carcinoembryonic antigen) (CEA) was prepared by using aminodextran (Mr=40 000) as the intermediate carrier, and the carbohydrate moiety of the antibody as the linking site. The resulting immunoconjugate was subjected to an in vitro evaluation for the internalization on the target cells (LoVo), and compared to that of unconjugated antibody, as well as the cellular uptake of unconjugated doxorubicin. The internalization was evaluated microscopically by following the translocation of the red fluorescence of doxorubicin and the green fluorescence of the fluorescein-isothiocyanate-labeled goat anti-(mouse Ig) antibody, which visualizes the location of the primary mouse antibody. Anti-CEA monoclonal antibody (NP-4) was found to internalize into LoVo cells. The immunoconjugate made with this antibody was similarly internalized, and the doxorubicin was found to distribute with the primary antibody. The cell surface and cytoplasm were the major compartments of their distribution. These results indicate that the drug molecules were indeed delivered into the cells by the antibody as an intact conjugate. Unconjugated doxorubicin, on the contrary, was quickly absorbed by the cells and concentrated in the nucleus within 30 min, and never showed a distribution in the cytoplasm or cell membrane as in the nucleus by this procedure. The intermediate drug conjugate, doxorubicin-dextran, did not show internalization. The internalization of NP-4 antibody (or the doxorubicin conjugate) was also confirmed by studying the intracellular catabolism of the cell-bound antibody (or conjugate). The release of the degraded antibody by the cells, as differentiated by trichloroacetic acid precipitation techniques, was considered an indication of internalization. Lysosomes were involved in the degradation, since the process was markedly inhibited in the presence of the lysosomal enzyme inhibitor, ammonium chloride.

Key words

Doxorubicin immunoconjugate Carcinoembryonic antigen conjugate Internalization 

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Lisa B. Shih
    • 1
  • David M. Goldenberg
    • 1
  • Hong Xuan
    • 1
  • Helen W. -Z. Lu
    • 1
  • M. Jules Mattes
    • 1
  • Thomas C. Hall
    • 1
  1. 1.Garden State Cancer Center and Center for Molecular Medicine and ImmunologyNewarkUSA

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