The effect of a monoclonal antibody coupled to ricin A chain-derived peptides on endothelial cells in vitro: Insights into toxin-mediated vascular damage

Immunotoxins (ITs) containing plant or bacterial toxins have a dose-limitin g toxicity of vascular leak syndrome (VLS) in humans. The active A chain of ricin toxin (RTA), other toxins, ribosome-inactivating proteins, and the V LS-inducing cytokine IL-2 contain the conserved sequence motif (x)D(y) wher e x = L, I, G, or V and y = V,L, or S. RTA-derived LDV-containing peptides attached to a monoclonal antibody, RFB4, induce endothelial cell (EC) damag e in vitro and vascular leak in two animal models in vivo. We have now inve stigated the mechanism(s) by which this occurs and have found that (1) the exposed D75 in the LDV sequence in RTA and the C-terminal flanking threonin e play critical roles in the ability of RFB4-conjugated RTA peptide to bind to and damage ECs and (2) the LDV sequence in RTA induces early manifestat ions of apoptosis in HUVECs by activating caspase-3. These data suggest tha t RTA-mediated inhibition of protein synthesis (due to its active site) and apoptosis (due to LDV) may be mediated by different portions of the RTA mo lecule. These results suggest that ITs prepared with RTA mutants containing alterations in LDVT may kill tumor cells in vivo in the absence of EC-medi ated VLS. (C) 2000 Academic Press.