Genetically Designing a More Potent Anti-Pancreatic Cancer Agent by Simultaneously Cotargeting Human Il-13 and Egf Receptors in a Mouse Xenograft Model

Abstract

Objective-Investigators are currently interested in the EGFR and IL-13R as potential targets in the development of new biologicals for pancreatic cancer. Attempts to develop successful agents have met with difficulty. Our novel approach was to simultaneously target these receptors with EGF and IL-13 cloned on the same bispecific single chain molecule with truncated diphtheria toxin (DT 390) to determine if co-targeting with DTEGF13 had any advantages. Design-Proliferation experiments were performed to measure the potency and selectivity of bispecific DTEGF13 and its monospecific counterparts against pancreatic cancer cell lines Panc-1 and MiaPaCa-2 in vitro. DTEGF13 was then administered intratumorally to nude mice with MiaPaCa-2 flank tumors to measure efficacy and toxicity (weight loss). Results-In vitro, bispecific DTEGF13 was 2,800-fold more toxic than monospecific DTEGF or DTIL13 against Panc-1. A similar enhancement was observed in vitro when MiaPaCa-2 pancreatic cancer cells or H2981-T3 lung adenocarcinoma cells were studied. DTEGF13 activity was blockable with recombinant EGF13. DTEGF13 was potent (IC 50 = 0.00017 nM) against MiaPaCa-2, receptor specific, and significantly inhibited MiaPaCa-2 tumor in nude mice (p<0.008). Conclusions-In vitro studies show that the presence of both ligands on the same bispecific molecule is responsible for the superior activity of DTEGF13. Intratumoral administration showed that DTEGF13 was highly effective in checking aggressive tumor progression in mice. Lack of weight loss in these mice indicated that the drug was tolerated and a therapeutic index exists in an "on target" model in which DTEGF13 is cross-reactive with native mouse receptors.

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

“…This is unknown, but we have shown subcutaneous injection of EGF-containing BLT result in dermatological injury (28), a finding in agreement with the known reactivity of EGF with EGFR in skin (29). Furthermore, monospecific EGFKDEL is highly toxic to mice indicating that the 70% homology of human and mouse EGF is sufficient for reactivity rendering the mouse at least a partial “ontarget” model.…”

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

“…Our laboratory has designed and published BLTs which simultaneously bind to dual independent receptors on the surface of tumor cells [6, 8, 9, 27, 29, 30, 35]. However, EGFATFKDEL is the first targeted toxin that simultaneously attacks tumor cells and the blood supply that feeds them.…”

“…Proteins were produced as described previously with some minor modifications to improve yield and purity [30]. Solubilization of partially purified inclusion bodies was carried out in guanidine hypochloride plus 50 mM dithierythritol in the refolding buffer to decrease protein aggregation.…”