A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature

Abstract

A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter Correspondence to: Daniel A. Vallera, valle001@umn.edu. NIH Public AccessAuthor Manuscript J Neurooncol. Author manuscript; available in PMC 2012 June 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR

“…This drug was highly efficacious in treatment of established glioma in rodent xenograft models [8]. Xenograft models are informative, but targeting human cells in “non-target” immunosuppressed mice (that do not bind human EGF and ATF) does not yield the same clinical investigative information as studies in a large animal “ontarget” models where the drug cross-reacts with native EGFR and uPAR.…”

“…We showed that canine HSA tumor-initiating cells express EGFR and uPAR, and that these cells are highly sensitive to eBAT [8,21–23]. Here, we used a large “ontarget” animal study that closely parallels what could be a human clinical trial to show feasibility, safety, and efficacy of eBAT to treat sarcomas in a clinically translatable setting using spontaneous canine HSA as model, in both naive disease and minimal residual disease settings.…”

A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

“…33 An impressive array of similar agents has been designed and tested using bispecific scFvs against EpCAM, various growth-factor and cytokine receptors, as well as an urokinase-type plasminogen activator receptor (uPAR), to import toxic cargo into various types of human cancer cells in culture and in mice xenografted with human cancer tissues. [34][35][36][37][38] For most of these agents, investigators have shown that the dualtargeting proteins had more potent anti-cancer activity than the corresponding mono-targeting control agents. We are not aware, however, of any studies attempting to demonstrate directly that the dual-targeting agent indeed preferentially eliminated antigen dp over sp cells when both were present in the same reaction mixture, mimicking the simultaneous presence of both types of cells in an infiltrated cancerous tissue.…”

A ligand-based and enediyne-energized bispecific fusion protein targeting epidermal growth factor receptor and insulin-like growth factor-1 receptor shows potent antitumor efficacy against esophageal cancer

“…Dual targeting strategies with bispecific agents have been classified into two types: i) those that directly act on target molecules, such as bispecific antibodies and ii) those that depend on targets for delivering an active moiety to killing tumor cells, such as bispecific immunotoxins or fusion proteins. A number of bispecific antibodies targeting both EGFR and IGF-1R (EI-04 and XGFR) have demonstrated superior antitumor activity in preclinical models (33,34), and bispecific immunotoxins/fusion proteins developed by Vallera et al also demonstrated either enhanced antitumor activity or broader spectrum of reactivity than the mono-specific molecules (35)(36)(37). EGF-LDP-IGF-AE is a bispecific enediyne-energized fusion protein that was constructed by fusing the natural ligands of EGFR and IGF-1R (EGF and IGF-1) to an enediyne antibiotic lidamycin (LDM; C1027) with potent antitumor activity.…”