Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.

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...

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Cis-diamminedichloroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran-avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (KB) cells over-expressing EGF-R. Cis-Pt-carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site.