Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use

Abstract

Purpose: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use.

Procedures: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 μg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively.

Results: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye.

Conclusion: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Keywords: ABY-029; Affibody; EGFR; Epidermal growth factor receptor; Fluorescence guided surgery; Glioma; IRDye 800CW.

Figure 1

In vitro phototoxicity of ABY-029 compared to BPD, a common photosensitizing agent. a F98, an EGFR(−) cell line, demonstrates phototoxicity to BPD but not ABY-029. b F98-EGFR, an EGFR(+) cell line, also demonstrates phototoxicity to BPD and not ABY-029. * = statistical significance from No Drug + 0 J/cm² group and No Drug within each light dose group, where p < 0.05. # = statistical significance from No Drug + 0 J/cm² group, where p < 0.05. There was no statistical significance between any F98 and F98-EGFR group.

Figure 2

The biodistribution of ABY-029 (2,449 µg/kg) in key organs one hour after intravenous injection as measured in the Pearl Imaging System. The values for male and female are presented separately and averaged together for all organs with the exception of the sex organs.

Figure 3

The pharmacokinetic clearance of 2449 µg/kg ABY-029 from the plasma of normal Sprague Dawley rats. The inset shows the log-log plot of the concentration of ABY-029 in blood for 0–720 minutes to visualize the short time periods more clearly.