IMMUNOTOXINS CONTAINING PSEUDOMONAS EXOTOXIN THAT TARGET LE(Y) DAMAGEHUMAN ENDOTHELIAL-CELLS IN AN ANTIBODY-SPECIFIC MODE - RELEVANCE TO VASCULAR LEAK SYNDROME

Vascular leak syndrome (VLS) was originally found to be a major dose-l imiting toxicity in humans with cancer treated with several immunotoxi ns (ITs) containing ricin A chain or blocked ricin, Recently, VLS has also been observed in patients treated with an IT containing the murin e monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant trunc ated form of Pseudomonas exotoxin (PE) A, Antibody B3 (IgG1k) recogniz es Lewis(Y) and related carbohydrate epitopes present on many human so lid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen, In the clinical tria l, the development of VLS has prevented the administration of the amou nt of IT necessary to achieve blood levels required for good therapeut ic responses, We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechan ism of VLS induced by ITs containing PE, To assess the cytotoxic effec t of IT on endothelial cells, various ITs were incubated with cells fo r 2 or 20 h, and the incorporation of [H-3]leucine into protein was me asured, The endothelial cells studied were human umbilical vein endoth elial cells, human lung-derived microvascular endothelial cells (HUVEC s), human adult dermal microvascular endothelial cells, human pulmonar y artery endothelial cells, and human aortic endothelial cells, We fou nd that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with P E38, as welt as B3(Fv)-PE38 (LMB-7), a recombinant single chain immuno toxin, 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 microv ascular endothelial cells but not on human pulmonary artery endothelia l cells, The cytotoxic effect was specific since PE38 itself or PE cou pled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml, Further evidence that the cytotoxici ty of B3-containing ITs is due to specific B3 binding to endothelial c ells comes from the fact that the cytotoxicity can be blocked by exces s free MAb B3. HUVECs undergo overt morphological changes after treatm ent with B3-LysPE38 or B3(Fv)PE38, Gaps between the cells are formed a fter 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 t o high concentrations of LMB-1.