Abstract 2858: Immuno-PET with site-specific labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake in a subcutaneous HER2 positive xenograft mouse model of ovarian adenocarcinoma

Abstract

Abstract Introduction: The combination of PET with the specificity of monoclonal antibodies (mAbs), immuno-PET, is an attractive approach to improve tumor detection and mAb quantification. 89Zirconium (89Zr) is particularly well-matched for the long circulatory half-life of intact mAbs. Previously, the use of 89Zr has been limited by the lack of suitable methods for stable coupling to mAbs. In addition, the conventional labeling strategies with random introduction of a chelator potentially reduce antibody binding and affinity towards target. Here we demonstrate the application of an enzyme- and click chemistry-mediated methodology for site-specifically labeled 89Zr immuno-PET imaging probes and compare them to a random labeled probe. Experimental procedures: Trastuzumab was conjugated to the p-SCN-Bn-Deferoxamine (p-SCN-Bn-DFO) chelator in three alternate ways: (1) randomly on lysine residues or site-specifically on enzymatically treated glycans using either (2) ß-galactosidase or (3) endoglycosidase S2. DFO-trastuzumab was radiolabeled with 89Zr (89Zr-DFO-trastuzumab) and injected into SK-OV-3 tumor-bearing NMRI nude mice. In addition, a dose-escalation study was performed with co-injection of either 100 µg or 500 µg of unlabeled trastuzumab. At 24, 70 and 110 hours post-injection, mice underwent small animal PET/CT imaging for longitudinal assessment. Mice were euthanized and organs resected for ex vivo biodistribution after the last imaging time-point. Additionally, the immuno-reactivity and tracer stability in buffer and plasma were assessed for all three tracers. Results: All tracers were found to be stable for up to 5 days post-labeling. PET imaging at 24, 70 and 110 hours after injection of 89Zr-DFO-trastuzumab revealed a gradual increase in tumor uptake and image contrast over the time-course. At 70 hours post-injection mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g), for random, ß-galactosidase or endoglycosidase S2 labeled probes, respectively. Site-specific labeling significantly increased tumor PET uptake (One-way ANOVA, p < 0.0001) at all time-points compared to random labeling. This was further supported by an immuno-reactivity of 93% for site-specific labeled probes compared to 80% for the random labeled trastuzumab. Titration with unlabeled trastuzumab did not affect tumor uptake considerably and the ex vivo biodistribution confirmed the data obtained by in vivo PET imaging. Conclusions: 89Zr-DFO-trastuzumab is well suited for specific immuno-PET imaging of HER2 positive ovarian cancer and site-specific labeling of trastuzumab presents with markedly higher immuno-reactivity and tumor specificity. These findings support the further development of site-specific radiolabeled monoclonal antibodies for immuno-PET. Citation Format: Lotte K. Kristensen, Camilla Christensen, Camilla S. Knudsen, Mette M. Jensen, Brian J. Agnew, Andreas Kjaer, Carsten H. Nielsen. Immuno-PET with site-specific labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake in a subcutaneous HER2 positive xenograft mouse model of ovarian adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2017-2858