DEVELOPMENT OF EXPERIMENTAL TARGETED TOXIN THERAPIES FOR MALIGNANT GLIOMA - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 309

bind to and kill human cells [18]. Domain II is the site of
proteolytic cleavage and is responsible for catalyzing trans-
location of the toxin into the cytosol. Domain III, located at
the C-terminus, possesses ADP-ribosylation activity, which
in turn leads to inactivation of EF-2, eventually leading to
cell death. In the genetically engineered PE molecule
PE38KDEL, amino acids (AA) 253-364 were linked to
AA 381-608, which in turn were fused to KDEL (an
endoplasmic reticulum retaining sequence) at position
609-612. To improve the binding of IL-4 toxin to the IL-
4R, a circularly permuted form of IL-4 was fused to the
toxin. This new agent was termed IL-4(38-37)-PE38KDEL
or IL-4-PE [34,35]. IL-4-PE was found to have a 16-fold
higher affinity for binding to GBM cell lines than the native
PE toxin, and was 3-30-fold more toxic to GBM cells
[36,37].
IL-4 is a pleiotropic cytokine composed of four a -helices,
which is primarily produced by Th 2 -type T lymphocytes
[38,39]. It has been suggested that residues on the A and C
helices bind to the IL-4R and are important for binding
affinity, whereas residues near the carboxy terminus bind to
a different subunit, presumably the common g chain of the
receptor, and are important for signal transduction [40,41].
However, the amino and carboxy termini are close together,
suggesting that attachment of a toxin moiety to the carboxy
terminus of IL-4 can disrupt receptor binding [18,34]. IL-4R
is a transmembrane protein composed of two subunits with
different functions, a 140-kDa chain and a 70-kDa g chain
[40]. IL-4R is also a component of the IL-13 receptor (IL-
13R) [41].
While normal CNS cells such as endothelial cells, micro-
glia, or astrocytes express low levels of IL-4R, malignant
glioma cells express high levels of this receptor on their
surface [23]. In one series, 16 of 21 surgical samples were
determined to express high numbers of IL-4R [19]. In
another series, all of the 25 tested surgical specimens of
high-grade gliomas expressed IL-4R. In addition, primary
cell cultures of glioma also expressed IL-4R, as determined
by RT-PCR and immunohistochemical analysis [42].
Another recent study demonstrated that 15 of 18 GBM
and 12 other brain tumor samples were moderately to
intensely positive for IL-4R. In contrast, no detectable IL-
4R was expressed in normal brain as determined by immu-
nohistochemistry [43].
Cell culture studies and animal models confirmed that IL-
4-PE is highly cytotoxic to immortalized GBM cell lines and
to primary glioma cell cultures [39,44]. In vivo efficacy was
evaluated in a nude mouse model with subcutaneously
injected human GBM cells [35]. Antitumor activity was
observed in 80% and 100% of animals treated with IL-4-PE
at 50 or 100 m g/kg for 3 days. To define the safety profile of
the toxin, IL-4-PE was administered intravenously (i.v.),
directly into the forebrain, and into the cisterna magna in
mice, rats, and nonhuman primates, respectively. In the

mouse, the approximate LD 50 following i.v. administration
was 475 m g/kg. At high doses ( > 400 m g/kg) hepatic necrosis
was seen. In the rat, direct injection into the forebrain of
275 ng/kg toxin caused no side effects, and in primates, the
maximum tolerated dose (MTD) following injection into the
cisterna magna was 6 m g/kg [35].
Weber et al. [45] carried out an open-label, dose-escala-
tion trial of intratumoral administration of IL-4-PE in
patients with recurrent malignant glioma. A total of 31 adult
patients were enrolled, 25 with GBM and 6 with anaplastic
astrocytoma. Patients were assigned to one of four groups
receiving different doses of toxin in different volumes of
infusate. Toxin was administered by CED via an external
pump connected to stereotactically placed catheters. The
overall median survival for the whole group of WHO grade 3
and 4 tumors was 8.2 months, with a median survival of 5.8
months for GBM patients. Six months survival was 52% and
48%, respectively. Drug-related grade 3 or 4 CNS toxicity
was seen in all groups in a total of 39% of all patients, and in
22% of patients at the MTD of 6 m g/mL in 40mL. Treat-
ment-related adverse events (AE) were limited to the CNS.
No deaths were attributable to treatment. No drug-related
systemic toxicity was apparent in any patient. Gadolinium-
enhanced MRI of the brain in some of the treated patients
showed areas of decreased signal intensity within the tumor
consistent with toxin-mediated tumor necrosis [45].
A multicenter, randomized, open-label Phase II study
with IL-4-PE was carried out in patients with recurrent GBM
to investigate continuous intratumoral infusion of the toxin
followed by surgical resection of tumor. The study was
designed to evaluate efficacy of IL-4-PE, with a secondary
objective to evaluate safety and tolerability of the toxin [46].
In the toxin group, patients received intratumoral infusion of
toxin at total doses of up to 90 m g and underwent surgical
resection of tumor between 2 and 7 days after the end of
toxin infusion. Patients in the control group underwent
tumor resection without prior toxin treatment. A total of
30 adult patients with unilateral, unifocal tumors with a
volume 60 have been enrolled.

Recruitment was completed in 2003, but no final published
results of the study are available yet [46]. There are currently
no Phase III protocols using IL-4-PE.

100mL and a KPS

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20.4.2 TP-38

TP-38 (Teva Pharmaceuticals, Miami, FL, formerly IVAX
Inc., Miami) is a recombinant chimeric protein molecule
composed of transforming growth factor a (TGF- a ), an
EGFR ligand, and the genetically engineered form of PE
described earlier (PE38KDEL).
The 170-180-kDa transmembrane glycoprotein, epider-
mal growth factor (EGF) receptor, is one of four members of
the ErbB family of receptor tyrosine kinases, which consist
of an extracellular domain that can bind ligands, a