Inhibition of human tumor growth in nude mice by a conjugate of doxorubicin with monoclonal antibodies to epidermal growth factor receptor.

Abstract

Monoclonal antibodies that recognize the extracellular domain of the epidermal growth factor receptor (mAblO8) were conjugated with doxorubicin through a dextran bridge. Several antibody-drug conjugates, containing different amounts of doxorubicin, retained binding capacity to human epidermoid carcinoma (KB) cells overexpressing epidermal growth factor receptors. Slight decrease in drug cytotoxicity was seen in in vitro tests, as determined either by inhibition of thymidine incorporation into cells or by reduction in number and size of KB-cell colonies. Yet, when tested in vivo against KB tumor xenografted into nude mice, the antiepidermal growth factor-receptor drug conjugates with high drug-substitution levels were significantly more effective than free doxorubicin, antibody alone, mixture of dextran-doxorubicin and antibody, or drug conjugated with irrelevant antibody. When the labile covalent bonds linking antibody to dextran bridge were stabilized by reduction, the therapeutic efficacy ofthe conjugate was markedly decreased. These results show that antibodies against the extracellular domain of the epidermal growth factor can deliver doxorubicin specifically and efficiently to tumor sites that express high receptor levels exerting a specific antitumor effect. (8).In the present study we examine the possibility of enhancing the antitumor effect of mAblO8 by conjugating it to an antineoplastic drug, doxorubicin. The drug was conjugated by means of periodate-oxidized dextran to the antibodies. We had previously shown that daunorubicin'conjugated to antibodies against rat a-fetoprotein was very effective in prolonging the survival time of intraperitoneally hepatomachallenged rats (9). This work was recently successfully extended to subcutaneous hepatoma tumors that metastasize to the lungs with or without surgical resection of'the'subcutaneous tumor (10). We report here results showing high efficacy of doxorubicin-anti-EGF-receptor conjugates in the treatment of subcutaneous xenografts of human KB cells in nude mice. The specific conjugate was significantly more effective than free drug, drug linked to an irrelevant antibody, and various other pertinent controls. MATERIALS AND METHODSAnimals. CD1 nude mice (aged 4-8 weeks) obtained from Charles River Breeding Laboratories were maintained at the Experimental Animals Center of the Weizmann Institute of Science.Cells. The KB human tumor cell line derived from oral epidermoid carcinoma was obtained from The American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (depleted of complement activity), glutamine, penicillin, streptomycin, and sodium pyruvate, at 370C in 5% C02/95% air.Antibodies. The mAblO8 IgG2a hybridoma line was generated by immunizing mice with Chinese hamster ovary cells expressing the extracellular domain of the human EGF receptor (11). The mAb were purified as described (8).Binding of Antibody to Doxorubicin. mAblO8 or antidinitrophenyl (DNP) mAb were linked to doxoru...

Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs

“…Some anti-HER-2 receptor antibodies act as partial agonists and promote weak or no activation of MAP kinase. As anti-HER-2 antibodies, tyrosine kinase inhibitors with speci®city for HER-2 kinase are also known to enhance the sensitivity of HER-2-overexpressing cancer cells to DNA-damaging agents (Aboud-Pirak et al, 1989;Tsai et al, 1996). Although the activity of rhuMAb HER-2 remains to be fully characterized, downstream eects of HER-2 stimulation, such as activation of MAP kinase, are likely to be aected by rhuMAb HER-2.…”

“…Prior work has shown some therapeutic advantage in the treatment of human tumors with anti-EGF receptor antibodies and doxorubicin (Aboud-Pirak et al, 1989), a drug commonly used in the treatment of breast cancer. Although anthracyclines are not generally considered to be DNA-damaging agents, recent work suggests these agents may elicit some indirect covalent modi®cations of DNA in mammary tissue (Purewal and Liehr, 1993).…”

“…Monoclonal antibodies to EGF receptor elicited an additive antitumor eect when given in combination with the anthracycline drug, doxorubicin (Aboud-Pirak et al, 1989). A poorly understood but probable synergistic eect between monoclonal antibodies to EGF receptor and the chemotherapy drug, cisplatin, has also been reported (Aboud-Pirak et al, 1988).…”

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

“…7,26,30,[135][136][137][138][139] Fourth, cytotoxic anti-neoplastic potency could have been delineated in vivo against human neoplastic xenographs in animal hosts as a model for human neoplasia where the efficacy of a covalent immunochemotherapeutic frequently tends to be higher than in ex vivo tissue culture based models utilizing the same identical cancer cell type. 39,42,140 The enhanced levels of covalent immunochemotherapeutic potency measured in vivo is presumed in part to be dependent upon contributions from endogenous immune responses that include antibody-dependent cell cytotoxicity (ADCC) phenomenon in concert with complemented-mediated cytolysis initiated by the formation of antigen-immunoglobulin complexes on the exterior surface membrane of ''targeted'' neoplastic cells. During ADCC events, the cell types that are actively involved in this response release cytotoxic components that are known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents.…”

MHC Class I–Related Chain A Conjugated to Antitumor Antibodies Can Sensitize Tumor Cells to Specific Lysis by Natural Killer Cells

“…However, despite encouraging results, the percentage of complete remission remains low and there is a definitive need to improve the capacity of these mAbs to induce cytotoxicity against targeted tumor cells, e.g., by mutating their Fc part or by coupling radioisotopes (1), drugs (2), or cytokines (3). To this end, we (4,5) and others (6, 7) recently described a new type of conjugate consisting of antitumor antibody FabV fragments chemically coupled or fused with soluble MHC class I molecules presenting antigenic viral peptides.…”