Abstract 4552: Pretargeted radioimmunoimaging and -therapy in tumor-bearing mice using a bioorthogonal reaction
Authors: Rossin Raffaella , Verkerk Pascal Renart , van den Bosch Sandra M. , Vulders Roland Cm , Verel Iris , Lub Johan , Robillard Marc S. 
Published: February 2011

Abstract
A major challenge of radioimmunotherapy (RIT) of cancer is to enhance the nuclear radiation dose delivered to the tumor while minimizing the dose in healthy tissues. Monoclonal antibodies (mAb) circulate for a long time and only slowly accumulate in the tumor, which, when used for RIT, leads to dose-limiting side effects in healthy organs. This efficacy-limiting factor can be circumvented by pretargeting, which involves tumor targeting of a mAb followed by binding of a small radiolabeled probe to the tumor-bound mAb. The superior image contrast and the ability to administer higher (therapeutic) radiation doses compared to directly labeled mAbs is offset by the drawbacks of the current biological pretargeting systems, involving either immunogenicity issues or extensive re-engineering of the parent mAb. To address this, we designed a novel pretargeting approach based on the bio-orthogonal chemical inverse-electron-demand Diels Alder reaction, employing a trans-cyclooctene-conjugated mAb and a radiolabeled tetrazine derivative, and we evaluated the in vitro stability and reactivity, and tumor targeting in mice bearing colorectal xenografts.
The in vitro stability and reactivity of 111In-labeled tetrazine and anti-TAG72 mAb CC49 functionalized with trans-cyclooctene (TCO) moieties through lysine residue conjugation were monitored in PBS, serum and blood. In vivo pretargeting was performed in LS174T-tumored mice using 125I-labeled CC49-TCO and 111In-labeled tetrazine and assessed by dual isotope biodistribution and SPECT imaging with a nanoSPECT/CT.
When 111In-tetrazine was administered to mice 1 day after CC49-TCO, the chemically-tagged tumors reacted rapidly with 111In-tetrazine, resulting in pronounced radioactivity localization throughout the tumor and good tumor contrast, as demonstrated by SPECT/CT imaging of live mice 3 h post injection: 4.18%ID/g, tumor-to-muscle ratio (T/M)=13.1. In mice treated with unmodified CC49, the tumor could not be discriminated from the surrounding tissue (0.28 %ID/g, T/M=0.5). Mice treated with TCO-modified rituximab, which lacks specificity for TAG72, showed the expected retention of 111In-tetrazine in blood and non-target organs, and a much reduced tumor accumulation (1.02 %ID/g, T/M=2.1). Corresponding biodistribution experiments revealed a remarkable 52-57% reaction yield between TCO and tetrazine moieties present in tumor and blood.
We have demonstrated the first use of a chemical reaction between two exogenous moieties in living animals for the non-invasive imaging of low-abundance targets in clinically relevant conditions. The inverse-electron-demand Diels Alder reaction has the potential to improve the state of the art of pretargeted RIT and can be applied to a range of antibodies due to its universal and straightforward conjugation chemistry. The validation for pre-targeted RIT in LS174T-tumored mice is underway.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4552.