Immunotoxins containing Pseudomonas exotoxin that target LeY damage human endothelial cells in an antibody-specific mode: relevance to vascular leak syndrome.
Vascular leak syndrome (VLS) was originally found to be a major dose-limiting toxicity in humans with cancer treated with several immunotoxins (ITs) containing ricin A chain or blocked ricin. Recently, VLS has also been observed in patients treated with an IT containing the murine monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant truncated form of Pseudomonas exotoxin (PE) A. Antibody B3 (IgG1k) recognizes LewisY and related carbohydrate epitopes present on many human solid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen. In the clinical trial, the development of VLS has prevented the administration of the amount of IT necessary to achieve blood levels required for good therapeutic responses. We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechanism of VLS induced by ITs containing PE. To assess the cytotoxic effect of IT on endothelial cells, various ITs were incubated with cells for 2 or 20 h, and the incorporation of [3H]leucine into protein was measured. The endothelial cells studied were human umbilical vein endothelial cells, human lung-derived microvascular endothelial cells (HUVECs), human adult dermal microvascular endothelial cells, human pulmonary artery endothelial cells, and human aortic endothelial cells. We found that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with PE38, as well as B3(Fv)-PE38 (LMB-7), a recombinant single chain immunotoxin, inhibited protein synthesis, with 50% inhibitory concentrations between 600 and 1000 ng/ml for 20-h incubation in HUVECs, human lung-derived microvascular endothelial cells, and human adult dermal microvascular endothelial cells but not on human pulmonary artery endothelial cells. The cytotoxic effect was specific since PE38 itself or PE coupled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml. Further evidence that the cytotoxicity of B3-containing ITs is due to specific B3 binding to endothelial cells comes from the fact that the cytotoxicity can be blocked by excess free MAb B3. HUVECs undergo overt morphological changes after treatment with B3-LysPE38 or B3(Fv)PE38. Gaps between the cells are formed after a 20-h exposure but not after 2 h. These studies suggest that VLS in patients is due to capillary damage caused by prolonged exposure to high concentrations of LMB-1.
Kuan
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CT
Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Pai
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LH
Pastan
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D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000009:adverse effects*; D000918:Antibody Specificity; D001427:Bacterial Toxins; D019559:Capillary Leak Syndrome / Q000209:etiology*; D002478:Cells, Cultured / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects*; D005098:Exotoxins / Q000009:adverse effects* / Q000737:chemistry; D006801:Humans; D000922:Immunotoxins / Q000009:adverse effects* / Q000737:chemistry; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D007983:Lewis Blood Group Antigens; D011506:Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C094162:Lewis Y antigen