Internalization of an intact doxorubicin immunoconjugate

Abstract

An immunoconjugate between doxorubicin and anti-(carcinoembryonic antigen) (CEA) was prepared by using aminodextran (M(r) = 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 (Lo Vo), 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 Lo Vo 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.

Synthesis of a Covalent Epirubicin-(C3-amide)-Anti-HER2/neuImmunochemotherapeutic Utilizing a UV-Photoactivated Anthracycline Intermediate

“…Second, maintaining maximum binding-avidity also directly facilitates efficient and more extensive internalization of epirubicin-(C 3 -amide)-[anti-HER2/neu] by mechanisms of ligand-induced receptor-mediated-endocytosis at HER2/neu overexpressed by mammary adenocarcinoma 7,26,30,120 and other neoplastic cell types. [121][122][123][124][125] Seemingly modest alterations in synthetic chemistry conditions, and elevations in chemotherapeutic molar-incorporation-indexes can profoundly influence the selective binding properties of immunoglobulin fractions. 29 Relatively higher anthracyclineimmunoglobulin molar-incorporation-indexes could have been attained during the synthesis of epirubicin-(C 3 -amide)-[anti-HER2/neu] utilizing succinimidyl 4,4-azipentanoate.…”

“…121 Although specific kinetic data for the tropic receptors HER2/neu and EGFR expressed by mammary adenocarcinoma is limited metastatic multiple myeloma is known to internalize and metabolize *8 · 10 6 molecules of anti-CD74 monoclonal antibody per day. .…”

“…7,26,29,30,43,121,128 Receptormediated-endocytosis at membrane-associated HER2/neu complexes in this fashion can promote elevations in intracellular chemotherapeutic concentrations that approach and exceed 8.5 · 43 to > 100 · fold greater 128 levels than are possible through processes of simple passive anthracycline diffusion. In this scenario, > 50% of selectively delivered anthracycline at 24 hours is intracellularly retained 43 where it becomes primarily associated with either membrane structures or it distributes more extensively throughout the cytosol environment.…”

“…In this scenario, > 50% of selectively delivered anthracycline at 24 hours is intracellularly retained 43 where it becomes primarily associated with either membrane structures or it distributes more extensively throughout the cytosol environment. 121,129 Conversely, ''free'' anthracycline following passive diffusion across the intact lipid bilayer of cell membranes is primarily detected complexed with nuclear DNA < 30 minutes after initial exposure 121 while anthracycline liberated from covalent anthracyclineimmunochemotherapeutics reportedly distributes to, and accumulates within the nucleus, mitochondria, and golgi compartments. 63 One of the primary objectives for the molecular design, synthesis, and evaluation of the cytotoxic potency of [i] 21,30,130,131 In doing so, mammary adenocarcinoma SKBr-3 is given an extended opportunity to continually internalize progressively more epirubicin-(C 3 -amide)-[anti-HER2/neu] by mechanisms of receptor-mediated-endocytosis.…”

Antibody Drug Conjugates: A Leap Ahead in Cancer Treatment

“…In addition to the mechanism of drug release, the specific site of conjugation, the potency of the drug and the average number of drug molecules per antibody needs to be carefully considered in the selection of the linker. Early ADCs incorporated drugs such as methotrexate [48][49][50] , vinblastine 51,52 and doxorubicin 11,18,53,54 each of which had displayed clinical activity as free drugs. In general these ADCs have demonstrated antigen-specific activity in vitro and in vivo but they required high dose levels of ADC to achieve substantial and appreciable antitumor activity.…”