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

• Published in: Clinical cancer research 2009-10, Vol.15 (19), p.6137-6147
• Main Authors: Oh, Seunguk, Stish, Brad J, Sachdev, Deepali, Chen, Hua, Dudek, Arkadiusz Z, Vallera, Daniel A
• Format: Article
• Language: English
• Subjects: Animals; breast cancer; Breast Neoplasms - drug therapy; Breast Neoplasms - immunology; Breast Neoplasms - pathology; Carcinoma - drug therapy; Carcinoma - immunology; Carcinoma - pathology; Drug Resistance, Neoplasm - drug effects; Drug Resistance, Neoplasm - immunology; EGF; EGFR; Epidermal Growth Factor - administration & dosage; Epidermal Growth Factor - genetics; Epidermal Growth Factor - immunology; Epidermal Growth Factor - therapeutic use; exotoxin; Female; Humans; IL-4; IL-4R; immunotoxin; Immunotoxins - genetics; Immunotoxins - immunology; Immunotoxins - therapeutic use; Lymphocyte Activation - drug effects; Lymphocyte Activation - genetics; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis, Site-Directed; Neoplasm Metastasis; nude mice; Pseudomonas; Receptors, Interleukin-4 - administration & dosage; Receptors, Interleukin-4 - genetics; Receptors, Interleukin-4 - immunology; Receptors, Interleukin-4 - therapeutic use; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - therapeutic use; toxicity; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.CCR-09-0696

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)