Abstract A86: Extracellular domain shedding influences specific tumor uptake and kinetics of EGFR PET tracer 89Zr-imgatuzumab
Authors: Pool Martin , Kol Arjan , Lub-de Hooge Marjolijn N. , Gerdes Christian A. , de Jong Steven , de Vries Elisabeth G.E. , Terwisscha van Scheltinga Anton G.T. 
Published: May 2016

Abstract
Background
Overexpression and mutations of epidermal growth factor receptor (EGFR) are associated with tumor cell growth, differentiation, proliferation, apoptosis and cellular invasiveness. Imgatuzumab is a novel EGFR monoclonal antibody (mAb), glycoengineered for enhanced antibody-dependent cellular cytotoxicity. Molecular imaging using radiolabeled mAbs can potentially support decision making during (pre)clinical development and clinical practice. However, preclinical EGFR imaging studies revealed a mismatch between in vivo EGFR expression levels and tumor tracer uptake. Factors suggested causing the mismatch include differences in perfusion rates, vascularity, vascular permeability, interstitial pressure and mAb plasma half-life. Another factor that might influence tracer kinetics is shed EGFR (sEGFR) extracellular domain (ECD), which is found in sera of cancer patients with EGFR expressing tumors. We radiolabeled imgatuzumab with zirconium-89 (89Zr) and determined the influence of sEGFR on 89Zr-imgatuzumab tracer kinetics and tumor uptake in xenograft models.
Methods
Imgatuzumab was conjugated to N-Suc-desferal and radiolabeled with 89Zr. MicroPET imaging was performed 24, 72 and 144 hours post injection of 10, 25 and 160 μg 89Zr-imgatuzumab (5 MBq). As a non-specific control, 111In-DTPA-IgG (1 MBq) was used in equal doses in the same animals. Imaging studies were performed in A431 (human epidermoid carcinoma, EGFR overexpressing) and A549 and H441 (both human non-small cell lung cancer, medium EGFR expressing) subcutaneous xenograft bearing mice. Ex vivo biodistribution analysis was performed after the last scan. sEGFR levels in liver lysates and plasma were obtained using a human EGFR ECD ELISA assay.
Results
Increasing 89Zr-imgatuzumab dose from 10 to 160 μg enhanced tumor uptake in A431 bearing mice from 8.7 ± 3.1 to 31.4 ± 11.6% ID/g. On contrary, dose escalation between 10 and 160 μg 89Zr-imgatuzumab lowered tumor uptake from 13.8 ± 5.9 to 6.7 ± 0.8% ID/g in A549 and from 27.6 ± 3.6 to 15.5 ± 3.2% ID/g in H441 bearing mice. High liver uptake of 22.0 ± 5.4% ID/g was observed in A431 tumors at 10 μg 89Zr-imgatuzumab, this was higher than A549 (7.4 ± 1.5% ID/g, p&lt;0.0001) and H441 (8.4 ± 2.1% ID/g, p&lt;0.001) bearing mice. Liver uptake in A431 bearing mice could be blocked with 160 μg 89Zr-imgatuzumab to 7.1 ± 1.6% ID/g. Human EGFR ECD ELISA showed presence of 790 ± 267 ng/g protein sEGFR in liver lysates and 831 ± 173 ng/ml in plasma of A431 bearing mice, while sEGFR levels in liver and plasma of A549 and H441 tumor bearing mice were comparable to non tumor bearing control mice.
Conclusion
89Zr-imgatuzumab effectively accumulates in EGFR expressing tumors. A431 tumors extensively shedded EGFR, which highly influenced 89Zr-imgatuzumab kinetics in A431 bearing mice. These results support the use of shed antigen measurements and subsequent tracer dose adjustment in future EGFR imaging studies.
Citation Format: Martin Pool, Arjan Kol, Marjolijn N. Lub-de Hooge, Christian A. Gerdes, Steven de Jong, Elisabeth G.E. de Vries, Anton G.T. Terwisscha van Scheltinga. Extracellular domain shedding influences specific tumor uptake and kinetics of EGFR PET tracer 89Zr-imgatuzumab. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A86.