Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain.
We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice. In the present experiments, we have attempted to improve the antitumor activity of monoclonal antibody 528 IgG2a against the EGF receptor by linking it to recombinant ricin A chain (rRA). The immunoconjugate (528 IgG-rRA) showed a potent cytotoxic effect on A431 cells in vitro. At a concentration of 10 pM, it inhibited the proliferation of cultured A431 cells by 50% and also inhibited protein synthesis in these cells by 50%. Proliferation was prevented and cell death occurred at 528 IgG-rRA concentrations of 60 pM or greater. Recombinant free ricin A chain was far less toxic. The cytotoxic effect of the immunoconjugate was neutralized by 528 IgG at concentrations 100-fold higher than 528 IgG-rRA. When the cytotoxic effect of 528 IgG-rRA was compared among several human cell lines expressing different numbers of EGF receptors, the capacity to inhibit both proliferation and protein synthesis generally correlated with the number of EGF receptors on the plasma membranes of these cells. Since 528 IgG-rRA is a very potent immunotoxin against A431 cells in culture, we designed experiments to test its in vivo antitumor activity against A431 xenografts in athymic mice. To measure the clearance of 528 IgG-rRA, 50 micrograms of immunotoxin were injected i.p. into athymic mice, blood was collected from the animals at regular intervals, and the level of immunotoxin in the serum was assayed by protein synthesis inhibition in cultured A431 cells. The blood level of active immunoconjugate reached a maximum 6 h after i.p. injection. The half-life of the absorption phase was 2.2 h, the half-life for elimination was 9.2 h, and blood levels which could be potentially cytotoxic were maintained for 48-72 h. We investigated a number of immunotoxin treatment schedules, including every other day for 4 days, based on these data. The results demonstrate that, while 528 IgG-rRA has higher in vivo antitumor activity than 528 IgG against A431 cell xenografts, this is accompanied by toxicity against the murine host.
Masui
H
H
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Kamrath
H
H
Apell
G
G
Houston
L L
LL
Mendelsohn
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000915:Antibody Affinity; D002294:Carcinoma, Squamous Cell / Q000628:therapy*; D002470:Cell Survival; D003602:Cytotoxicity, Immunologic; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy; D000922:Immunotoxins / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009368:Neoplasm Transplantation; D012276:Ricin / Q000008:administration & dosage*; D014407:Tumor Cells, Cultured / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D009363:Neoplasm Proteins; D012276:Ricin; D066246:ErbB Receptors