About 40% of malignant gliomas have amplification of the epidermal growth factor receptor (EGFR) gene. Chimeric proteins composed of epidermal growth factor linked to bacterial exotoxins kill such tumor cells in- vitro via receptor-mediated endocytosis. Unfortunately, systemic administration of these agents leads to severe toxicity. Moreover, since penetration of the blood-brain barrier by systemically administered fusion proteins is poor, other means od delivery such as interstitial therapy may be preferable. The studies proposed here are designed to examine the in-vitro tumor cell-killing effect of a variety of cytotoxic fusion proteins in which EGF is linked to a portion of the Pseudomonas exotoxin A-chain (PE). First, we will construct a plasmic in which the factor Xa cleavage recognition site is interposed between EGF and PE. Fusion proteins derived from this plasmid will be compared to other non- cleavable fusion proteins in which PE is positioned either at the N- or C-terminus of EGF. Proteins will be assessed with respect to their cell killing potency, catalytic activity, and ability to inhibit protein synthesis. Second, we will clone a series of astrocytoma cell lines derived from human tumor explants that are resistant to the effects of EGF-PE fusion proteins. Clones which continue to express all or a portion of the EGFR will be evaluated in detail. Third, we will develop C6 glioma cell lines which stably express wild type EGFR and later mutant EGFRs cloned from resistant subpopulations of human glioma explants. These cell lines will be used to assess the ability of such mutant receptors to bind fusion proteins. We believe that these studies will provide insights into the basis of EGF-EGFR interaction as well as the potential utility of cytotoxic fusion proteins in future brain tumor therapies. Moreover, the studies outlined here will create the tools necessary to begin to assess in-vivo responsiveness of solid brain tumors to such agents in immunocompetent hosts.
