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

Abstract

Purpose: To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE 38 KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. Experimental Design: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE 38 ). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. Results: EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by ∼90% with no apparent loss in in vitro or in vivo activity. Conclusions: Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials. (Clin Cancer Res 2009;15(19):6137-47)

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

“…Furthermore, using H2373 cells stably expressing green fluorescent protein for our in vivo study, we were able to accurately assess and quantitate peritoneal tumour colonies at the completion of treatment. This strategy has been shown to be much more sensitive for detecting and assessing tumour nodules (Udagawa et al, 2002;Oh et al, 2009). …”

“…Additional studies were performed and anti-EGFR or IL-4R antibodies were used to block EGFRKDEL activity. Each partially blocked (Oh et al, 2009). These results show that both the EGF and IL-4 ligands specifically target their respective receptors and each ligand contributes individually to the binding and subsequent cytotoxicity of EGF4KDEL.…”

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.…”

Targeting a Cancer-Specific Epitope of the Epidermal Growth Factor Receptor in Triple-Negative Breast Cancer

“…Ligandbased toxins with TGFa and EGF have been developed previously (49). Early examples included TGFa-PE40 (29) and DAB389EGF/486EGF (50) while more recent studies have explored dual targeting to EGFR and cytokine receptors (51). While these agents are considered suitable for local administration for treatment of glioblastomas or for intratumoral injections, systemic toxicity precludes wider use, underlining the importance of future development and use of conformation-dependent antibodies, such as 806, to target cancer-expressed EGFRs.…”