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.
OBJECTIVE
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.


METHODS
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 approximately 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.
Oh
Seunguk
S
Masonic Cancer Center, Section on Molecular Cancer Therapeutics, Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Stish
Brad J
BJ
Sachdev
Deepali
D
Chen
Hua
H
Dudek
Arkadiusz Z
AZ
Vallera
Daniel A
DA
D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D002277:Carcinoma / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000276:immunology; D004815:Epidermal Growth Factor / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016297:Mutagenesis, Site-Directed; D009362:Neoplasm Metastasis; D019948:Receptors, Interleukin-4 / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000627:therapeutic use*; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D000922:Immunotoxins; D019948:Receptors, Interleukin-4; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor