CYTOTOXICITY OF FOLATE-PSEUDOMONAS EXOTOXIN CONJUGATES TOWARD TUMOR-CELLS - CONTRIBUTION OF TRANSLOCATION DOMAIN

Folate-protein conjugates can be nondestructively delivered into a cel l's cytoplasm via folate receptor-mediated endocytosis if (i) the targ et cells express a folate-binding protein, and (ii) if the folate is l inked to its attached protein at a site that does not interfere with r eceptor recognition. Because such conjugates have been observed to rem ain in endosomal compartments for extended periods following cellular uptake, we decided to evaluate whether release into the cytoplasm migh t be expedited by inclusion of a translocation domain in the folate-pr otein construct. To test this possibility, momordin-folate and truncat ed Pseudomonas exotoxin-folate conjugates (LysPE38 and CysPE35), i.e. protein synthesis inhibitors either lacking or containing the desired translocation domain, respectively, were examined for their abilities to block protein synthesis in a variety of cell types. The translocati on competent LysPE38-folate construct was found to kill cells six time s more rapidly with 10-fold greater potency than the permeation-incomp etent momordin-folate. Further, cells expressing low levels of folate receptors could only be exterminated by the translocation competent Ps eudomonas exotoxin-folate conjugates. When the translocation capabilit y of CysPE35-folate was inactivated by modification of Cys287, the con struct also lost most of its cytotoxicity. These data suggest that aut ocatalysis of transport from an internal vesicular compartment into th e cytoplasm can greatly augment the cytotoxicity of a protein toxin en tering cells via the folate endocytosis pathway. Because the folate li gand can selectively target a protein conjugate to cancer cells in the presence of normal cells, such translocatable toxin-folate constructs warrant further study as a possible treatment for some malignancies.