The authors of this study aimed to genetically design a bispecific targeted toxin that would simultaneously target overexpressed markers on glioma as well as the tumor vasculature, to mutate certain amino acids to reduce the immunogenicity of this new drug, and to determine whether the drug was able to effectively reduce aggressive human brain tumors in a rat xenograft model via a novel hollow fiber (HF) catheter delivery system.
A new bispecific ligand-directed toxin (BLT) was created in which 2 human cytokines—epidermal growth factor ([EGF], targeting overexpressed EGF receptor) and amino acid terminal fragment ([ATF], targeting urokinase plasminogen activator receptor)—were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin with a terminal lysyl-aspartyl-glutamyl-leucine (KDEL) sequence. Site-specific mutagenesis was used to mutate amino acids in 7 key epitopic toxin regions that dictate the B cell generation of neutralizing antitoxin antibodies to deimmunize the drug, now called “EGFATFKDEL 7mut.” Bioassays were used to determine whether mutation reduced the drug's potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were decreased. Aggressive brain tumors were intracranially established in nude rats by using human U87 glioma genetically marked with a firefly luciferase reporter gene (U87-luc), and the rats were stereotactically treated with 2 intracranial injections of deimmunized EGFATFKDEL via convection-enhanced delivery (CED). Drug was administered through a novel HF catheter to reduce drug backflow upon delivery.
In vitro, EGFATFKDEL 7mut selectively killed the human glioblastoma cell line U87-luc as well as cultured human endothelial cells in the form of the human umbilical vein endothelial cells. Deimmunization did not reduce drug activity. In vivo, when rats with brain tumors were intracranially treated with drug via CED and a novel HF catheter to reduce backflow, there were significant tumor reductions in 2 experiments (p < 0.01). Some rats survived with a tumor-free status until 130 days post–tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The maximal tolerated dose of EGFATFKDEL 7mut was established at 2 μg/injection or 8.0 μg/kg, and data indicated that this dose was nontoxic. Antitoxin antibodies were reduced by at least 90%.
First, data indicated that the BLT framework is effective for simultaneously targeting glioma and its neovasculature. Second, in the rodent CED studies, newly developed HF catheters that limit backflow are effective for drug delivery. Third, by mutating critical amino acids, the authors reduced the threat of the interference of neutralizing antibodies that are generated against the drug. The authors' experiments addressed some of the most urgent limitations in the targeted toxin field.
HassanRBullockSPremkumarAKreitmanRJKindlerHWillinghamMC: Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res13:5144–51492007
LiuTFHallPDCohenKAWillinghamMCCaiJThorburnA: Interstitial diphtheria toxin-epidermal growth factor fusion protein therapy produces regressions of subcutaneous human glioblastoma multiforme tumors in athymic nude mice. Clin Cancer Res11:329–3342005
LiuTFTatterSBWillinghamMCYangMHuJJFrankelAE: Growth factor receptor expression varies among high-grade gliomas and normal brain: epidermal growth factor receptor has excellent properties for interstitial fusion protein therapy. Mol Cancer Ther2:783–7872003
LiuTFWillinghamMCTatterSBCohenKALoweACThorburnA: Diphtheria toxin-epidermal growth factor fusion protein and Pseudomonas exotoxin-interleukin 13 fusion protein exert synergistic toxicity against human glioblastoma multiforme cells. Bioconjug Chem14:1107–11142003
MoriTAbeTWakabayashiYHikawaTMatsuoKYamadaY: Up-regulation of urokinase-type plasminogen activator and its receptor correlates with enhanced invasion activity of human glioma cells mediated by transforming growth factor-α or basic fibroblast growth factor. J Neurooncol46:115–1232000
OhSOhlfestJRTodhunterDAValleraVDHallWAChenH: Intracranial elimination of human glioblastoma brain tumors in nude rats using the bispecific ligand-directed toxin, DTEGF13 and convection enhanced delivery. J Neurooncol95:331–3422009
OhSStishBJSachdevDChenHDudekAZValleraDA: A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma. Clin Cancer Res15:6137–61472009
OndaMNagataSFitzGeraldDJBeersRFisherRJVincentJJ: Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients. J Immunol177:8822–88342006
ParneyIFKunwarSMcDermottMBergerMPradosMChaS: Neuroradiographic changes following convection-enhanced delivery of the recombinant cytotoxin interleukin 13-PE38QQR for recurrent malignant glioma. J Neurosurg102:267–2752005
RamZBarnettGVogelbaumMConstantiniSLilleheiKShermanJ: Pre-operative infusion of IL13-PE38QQR cytotoxin by convection-enhanced delivery (CED) in recurrent malignant glioma: a phase I/II study. 2003 Proceedings of the American Society of Clinical Oncology Annual MeetingAlexandria, VAASCO2003. Abstract #403
RamakrishnanSOlsonTABautchVLMohanrajD: Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo. Cancer Res56:1324–13301996
RustamzadehEValleraDATodhunterDALowWCPanoskaltsis-MortariAHallWA: Immunotoxin pharmacokinetics: a comparison of the anti-glioblastoma bi-specific fusion protein (DTAT13) to DTAT and DTIL13. J Neurooncol77:257–2662006
ShenGLLiJLVitettaES: Bispecific anti-CD22/anti-CD3-ricin A chain immunotoxin is cytotoxic to Daudi lymphoma cells but not T cells in vitro and shows both A-chain-mediated and LAK-T-mediated killing. J Immunol152:2368–23761994
StishBJOhSChenHDudekAZKratzkeRAValleraDA: Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity. Br J Cancer101:1114–11232009
TodhunterDAHallWARustamzadehEShuYDoumbiaSOValleraDA: A bispecific immunotoxin (DTAT13) targeting human IL-13 receptor (IL-13R) and urokinase-type plasminogen activator receptor (uPAR) in a mouse xenograft model. Protein Eng Des Sel17:157–1642004
TsaiAKOhSChenHShuYOhlfestJRValleraDA: A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature. J Neurooncol[epub ahead of print]2010
ValleraDATodhunterDAKurokiDWShuYSichenederAChenH: A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin Cancer Res11:3879–38882005
ValleraDATodhunterDKurokiDWShuYSichenederAPanoskaltsis-MortariA: Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice. Leuk Res29:331–3412005
VogelbaumMASampsonJHKunwarSChangSMShaffreyMAsherAL: Convection-enhanced delivery of cintredekin besudotox (interleukin-13-PE38QQR) followed by radiation therapy with and without temozolomide in newly diagnosed malignant gliomas: phase 1 study of final safety results. Neurosurgery61:1031–10382007