HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors

Abstract

Purpose Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. Experimental Design The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega, Madison, WI). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancers, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. Results In vitro experiments showed that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. Conclusions Our findings demonstrated that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

“…Recently, we demonstrated for the first time the compatibility of the DARPin format with PE40 (here denoted ETA 00 ) to produce high yields of a potent anti-EpCAM fusion toxin (25). Because, however, these small recombinant proteins have an elimination half-life of hardly more than 10 minutes (25,30,31), which limits tumor localization, pharmacologic improvements are mandatory. PEGylation offers several advantages for biomedical compounds, including an increased hydrodynamic radius and serum stability, resulting in increased blood residence time due to decreased proteolysis, renal filtration, and liver clearance, and delayed recognition by the immune system (32,33).…”

New developments in the treatment of HER2-positive breast cancer

“…Liver enzyme tests on mice treated with HER2-affitoxin showed that the average alanine aminotransferase plasma level was three times higher than in control animals but that the aspartate aminotransferase ratio was less than 2. 115 No significant differences in alkaline phosphatase or bilirubin levels were observed.…”

“…The amino acid sequence of an affibody molecule is based on a portion of the Staphylococcus aureus protein A. Production of affitoxin was performed by Zielinski et al 114,115 by subcloning the PE38 toxin sequence into a HER2-affibody expression construct, transforming bacteria with the construct, and inducing expression of affitoxin by stimulating with isopropyl-beta-D-thiogalactopyranoside. Nickel-affinity (HisTrap HP; GE Healthcare, Hertfordshire, UK) and anion exchange columns were used to purify affitoxin from cell lysates.…”

“…115 HER2-targeted affitoxin (1.5 mg/kg), a control non-HER2-targeted affitoxin, or saline was delivered by intravenous injection every 3 days for a total of 24 days to athymic mice with BT474 xenografts. In contrast to the controls, HER2-affitoxin caused rapid tumor shrinkage of approximately 60% of the initial size within the first 3 days of treatment.…”

Learning from the past: historical aspects of bacterial toxins as pharmaceuticals

“…In another approach, the immune factor for the SPN toxin produced by Streptococcus pyogenes was suggested as a potential novel bactericidal agent [38]. An additional example is the recently engineered and promising anticancer agent derived by combining a modified Pseudomonas aeruginosa exotoxin A with a HER2-specific affibody, termed affitoxin [39]. Streptokinase, which is produced by streptococci, has been used to unclog arteries in patients suffering from stroke [40,41].…”