⁸⁹Zr-nimotuzumab for potential clinical translation as an anti-EGFR immunoPET agent

Objectives: Mutations that lead to epidermal growth factor receptor (EGFR) upregulation have been associated with a number of cancers, including glioblastoma, colon cancers, squamous-cell carcinoma of the lung and epithelial tumors of the head and neck. These somatic mutations involving EGFR lead to its constant activation which produces uncontrolled cell division, a hallmark of cancer. Nimotuzumab (Nz), a humanized monoclonal antibody (mAb) with an orphan drug status in the US and EU for glioma, binds to the EGFR. This binding block EGFR and stop the uncontrolled cell division. The objective of this study was to produce and characterize ⁸⁹Zr-desferoxamine-nimotuzumab (⁸⁹Zr-Df-Nz) and evaluate the pharmacokinetics, biodistribution, microPET imaging, radiation dosimetry and acute toxicity of ⁸⁹Zr-Df-Nz in tumor and non-tumor bearing mice in order to obtain regulatory approval to advance this agent to a first-in-humans Phase I/II clinical trial.

Methods: Nimotuzumab was conjugated with p-isothiocyanatobenzyl-desferioxamine (Df-Bz-NCS) and was consequently radiolabeled with Zr-89. Nimotuzumab and its immunoconjugate (Nz-Df) were characterized by mass spectroscopy, gel-electrophoresis, biolayer interferometry (BLI) and flow cytometry. The radioimmunoconjugate (⁸⁹Zr-Df-Nz) was characterized by in vitro saturation binding assay and evaluated in in vivo biodistribution study and microPET imaging in nude mice EGFR expressing xenografts (colorectal cancer: DLD-1 and breast cancer: MDA-MB-468) . The radiation dosimetry and toxicity of ⁸⁹Zr-Df-Nz was also evaluated in non-tumor bearing mice.

Results: In vitro experiments showed that both Nz and Nz-Df have high affinity for the EGFR receptor with K_D value of less than 10 nM. ⁸⁹Zr-Df-Nz showed saturation and high specific binding in EGFR expressing cell line (DLD-1) with a K_D value 14.1±2.6 nM. ⁸⁹Zr-Df-Nz exhibited bi-exponential elimination from the blood in non-tumor bearing mice with a distribution half-life (α-phase) of 0.2 h and an elimination phase half-life (β-phase) of 23.5 h. In vivo experiments in mice inoculated with two different xenograft models (DLD-1 and MDA-MB-648) showed that, ⁸⁹Zr-Df-Nz accumulates over time specifically in tumors and showed a high tumor-to-background contrast. Tumor uptake of ⁸⁹Zr-Df-Nz was 11.3±2.5 and 13.1±3.2 % injected dose per gram at 168 h post-injection in mice with DLD-1 and MDA-MB-468 xenograft, respectively. The radiation absorbed dose estimates predicted for humans from intravenous administration of ⁸⁹Zr-Df-Nz to mice showed that the organs that would receive the highest radiation absorbed doses are kidney, liver, spleen and lungs.

Conclusion: The manufacturing of ⁸⁹Zr-Df-Nz met the specifications required of radiopharmaceuticals for human use and preclinical studies predicted that ⁸⁹Zr-Df-Nimotuzumab would be safe to administer to humans at a dose of 148 MBq (4 mg). The radiopharmaceutical exhibited preclinical pharmacokinetic, biodistribution and radiation dosimetry properties suitable for advancement to a first-in-humans clinical trial. Research Support: Western Economic Diversification, Saskatchewan, Canada