We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529 -531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second ␣ helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. The transforming properties of HPVs reside in two genes, E6 and E7. The E6 and E7 genes are consistently expressed in HPV-positive cervical cancers and derived cell lines (5-7). They cooperate to immortalize primary human keratinocytes (8 -13). HPV-16 E6 also cooperates with activated Ras in the transformation and immortalization of baby mouse kidney cells and baby rat kidney cells (14, 15). Independently of E7 or ras, HPV-16 E6 can transform NIH 3T3 cells (16), immortalize human mammary epithelial cells (17), and induce keratinocyte resistance to calcium and serum-induced differentiation (18). The activity of E6 in different biological assays implies it may influence diverse cellular pathways.The ability of E6 protein to associate with the cellular tumor suppressor p53 has been suggested as the mechanism by which the viral protein promotes cell growth and proliferation (19). Although binding of high risk HPV E6s with p53 appears to be mediated by another cellular protein, E6AP (20), direct in vitro association of E6 with p53 has also been observed (21,22). The complex of E6 and E6AP functions as an ubiquitin-protein ligase that results in the specific ubiquitination and subsequent degradation of p53 (23). Accumulating evidence suggests that E6 has functions independent of inactivating p53 in cellular transformation (24 -36).We have recently identified a cDNA encoding a cellular protein that binds papillomavirus E6 (E6BP or ERC-55) (1). E6BP was identified as a calcium-binding protein of the endoplasmic reticulum (37). The localization of E6BP is consistent with the localization of E6 to nonnuclear membranes (38). In vitro binding experiments demonstrated that E6BP interacted specifically with E6 proteins from cancer-related HPV types and the bovine papillomavirus type 1 (BPV-1). The transforming activity of a set of previously characterized BPV-1 E6 mutants correlated well with their E6BP binding ability. These results suggest that the E6BP interaction plays an important role for BPV-1 E6-induced transformation. More recently, it was reported that BPV-1 E6 associated with paxillin (39) as well as the trans-Golgi network-specific clathrin adaptor complex AP-1 (40) and that E6 proteins from some high risk HPVs interacts wit...

A bispecific immunotoxin (IT) called DTAT13 was synthesized in order to target simultaneously the urokinase-type plasminogen activator receptor (uPAR)-expressing tumor neovasculature and IL-13 receptor expressing glioblastoma cells with the goal of intratumoral administration for brain tumors. The recombinant hybrid was created using the non-internalizing N-terminal fragment (ATF) of uPA and the IL-13 molecule for binding plus the catalytic and translocation portion of diphtheria toxin (DT) for killing. The 71 kDa protein was highly selective for human glioblastoma in vitro showing no loss on binding compared with DTAT and DTIL13 controls. In vivo, DTAT13 caused the regression of small tumors when administered at 10 micro g/day given on a five-dose schedule every other day. DTAT13 was able to target both overexpressed uPAR and the vasculature, as demonstrated by its ability to kill HUVEC cells. Also, mortality studies indicated that DTAT13 was less toxic than DTAT or DTIL13. These findings indicate that bispecific IT may allow treatment of a broader subset of antigenically diverse patients while simultaneously reducing the exposure to toxin required than if two separate agents were employed.

The poor prognosis associated with the current management of malignant gliomas has led investigators to develop alternative treatments such as targeted toxin therapy. The optimal method for administering these agents is under development but appears to be convection-enhanced delivery (CED). The direct intratumoral infusion of targeted toxins was first performed in nude mouse flank tumor models of human malignant glioma. After the demonstration of in vivo efficacy, these potent cytotoxic compounds were tested in Phase I and Phase II clinical trials. Using a high-flow microinfusion technique, volumes of up to 180 ml were infused by CED through catheters placed directly into brain tumors. Minor systemic toxicity was seen in the form of hepatic enzyme elevation. Neural toxicity manifested as seizure activity and hemiparesis resulted from peritumoral edema that followed the completion of the infusion. Peritumoral toxicity was believed to be more related to the concentration of the infused immunotoxin than to the infusion volume. In approximately half of patients treated with CED a stable disease course, a partial response, or a complete response was demonstrated in some clinical trials. Targeted toxin therapy has clinical efficacy in patients with malignant gliomas. Convection-enhanced delivery appears to represent an effective method for administering these agents in patients with malignant brain tumors.

A fusion protein consisting of human interleukin-13 and the first 389 amino acids of diphtheria toxin was assembled in order to target human glioblastoma cell lines in a murine intracranial model. In vitro studies to determine specificity indicated that the protein called DTIL13 was highly selective for human glioblastoma. In vivo, the maximum tolerated dose of DTIL13 was 1 lg/injection given every other day and repeated for 3 days. Doses that exceeded this amount resulted in weight loss and liver damage as determined by histology and enzyme assay. Experiments in IL-4 receptor knockout mice revealed that liver toxicity was receptorrelated. This same dose given to nude mice with established U373 MG brain tumors resulted in significant reductions in tumor volume and significantly prolonged survival (p < 0.0001). Magnetic resonance imaging (MRI) proved to be extremely useful in (i) determining the ability of DTIL13 to reduce tumor size and (ii) for studying toxicity since diffusion-weighted and gradient echoweighted MRI revealed that vascular leak syndrome was not a limiting toxicity at this dose. These results suggest that DTIL13 is as effective in an intracranial rodent model as it was in a flank model in previous studies and that DTIL13 might be an effective treatment for glioblastoma multiforme. ' 2005 Wiley-Liss, Inc.Key words: diphtheria toxin; glioblastoma; interleukin-13; brain tumor; immunotoxin Glioblastoma multiforme (GBM) is an incurable, heterogeneous, high-grade astrocytic glioma, thought to originate from glial nonneuronal cells. 1 More recent studies have identified a subpopulation of cancer stem-cells within GBM that retain the capacity for self-renewal and the ability to differentiate into a phenotypically diverse population of cells. 2-4 With a 2-year-patient survival rate of <30%, 5 effective treatment has been hindered by the lack of tumor-specific markers in the majority of patients. Recently, the IL-13 receptor (IL-13R) has been found to be overexpressed on cultured human GBM cell lines and surgical GBM specimens, but is not detectable in normal brain tissue. [6][7][8] Targeting the IL-13 receptor with an immunotoxin (IT) has already proven to have potential for treating brain tumors. [8][9][10][11][12] ITs are not mainstream pharmaceuticals, and treatment of systemic tumors with ITs has been limited by their failure to localize in tumor. The promise of these drugs is considerably greater for brain-cancer therapy, since IT can be directly administered intracranially. In an earlier study, we assembled an IL-13 IT made by fusing gene fragments encoding for human IL-13 and the first 389 amino acids of diphtheria toxin. 13 In vivo studies with this agent showed that it had a potent antitumor effect against IL-13R-positive GBM cells grown in the flanks of nude mice without significant toxicity.Although encouraging, the flank model has limitations as a model for intracranial therapy. First, intratumoral pressure within the flank model does not reach the pressures that occur in intracranial tumors ...

DTAT13, a novel recombinant bispecific immunotoxin (IT) consisting of truncated diphtheria toxin, an amino-terminal (AT) fragment of the urokinase-type plasminogen activator (uPa), and a fragment of human IL-13 was assembled in order to target receptors on glioblastoma multiforme (GBM) and its associated neovasculature. Previous in vitro studies confirmed the efficacy of DTAT13 against various GBM cell lines expressing both IL-13 receptor or uPA receptor, and previous in vivo testing demonstrated the efficacy of DTAT13 in significantly inhibiting a range of xenograft tumors and showed that DTAT13 was 160- and 8-fold less toxic to the parental fusion IT, DTAT and DTIL13, respectively. To further understand the properties of DTAT13, pharmacokinetic/biodistribution experiments were performed. Binding analysis revealed that the IL-13 domain functioned independently of the uPA domain and that the K (d) for each binding domain was essentially the same as that of DTIL13 and DTAT. Flow cytometry studies indicated that DTAT13 bound better than DTAT or DTIL13. Analysis of the rate of protein synthesis inhibition in U87 MG cells by DTAT13 compared to DTAT revealed a faster rate of inhibition with DTAT13 compared to DTAT. The rate of protein synthesis inhibition of DTAT13 was identical to that of DTIL13 in U373 MG cells. Intracranial biodistribution studies revealed that DTAT13 was able to cross to the contralateral hemisphere unlike DTIL13 but similar to DTAT. These studies show that DTAT13 has properties encompassing those of both DTIL13 and DTAT and warrants further consideration for clinical development.