Sandra M. van den Bosch

Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance.

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin and [Gd(HPDO3A)(H(2)O)] from different temperature-sensitive liposomes for applications in temperature-induced drug delivery under magnetic resonance image guidance. In particular, two temperature-sensitive systems composed of DPPC:MPPC:DPPE-PEG2000 (low temperature-sensitive liposomes, LTSL) and DPPC:HSPC:cholesterol:DPPE-PEG2000 (traditional temperature-sensitive liposomes, TTSL) were investigated. The co-encapsulation of [Gd(HPDO3A)(H(2)O)], a clinically approved MRI contrast agent, did not influence the encapsulation and release of doxorubicin. The LTSL system showed a higher leakage of doxorubicin at 37 degrees C, but a faster release of doxorubicin at 42 degrees C compared to the TTSL system. Furthermore, the rapid release of both doxorubicin and the MRI contrast agent from the liposomes occurred near the melting phase transition temperature, making it possible to image the release of doxorubicin using MRI.

Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.

One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biological capturing systems. A bioorthogonal chemical approach may circumvent this drawback, but effective in vivo chemistry in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels-Alder cycloaddition between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k2 = 2.7 × 10(5) M(-1) s(-1)) via the trans-cyclooctene, approaching the speed of biological interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-containing proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.

Block-copolymer-stabilized iodinated emulsions for use as CT contrast agents

The objective of this study was to develop radiopaque iodinated emulsions for use as CT blood pool contrast agents. Three hydrophobic iodinated oils were synthesized based on the 2,3,5-triiodobenzoate moiety and formulated into emulsions using either phospholipids or amphiphilic polymers, i.e. Pluronic F68 and poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO), as emulsifiers. The size, stability and cell viability was investigated for all stabilized emulsions. Three emulsions stabilized with either lipids or PBD-PEO were subsequently tested in vivo as a CT blood pool contrast agent in mice. While the lipid-stabilized emulsions turned out unstable in vivo, polymer-stabilized emulsions performed well in vivo. In blood, a contrast enhancement of 220 Hounsfield Units (HU) was measured directly after intravenous administration of 520 mg I/kg. The blood circulation half-life of a PBD-PEO stabilized emulsion was approximately 3 h and no noticeable in vivo toxicity was observed. These results show the potential of above emulsions for use as blood pool agents in contrast enhanced CT imaging.

Diels–Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody

Current pretargeting systems use noncovalent biologic interactions, which are prone to immunogenicity. We previously developed a novel approach based on the bioorthogonal reaction between a radiolabeled tetrazine and an antibody-conjugated trans-cyclooctene (TCO). However, the tumor-to-blood ratio was low due to reaction with freely circulating antibody-TCO. Methods: Here we developed 2 tetrazine-functionalized clearing agents that enable rapid reaction with and removal of a TCO-tagged antibody (CC49) from blood. Next, we incorporated this approach into an optimized pretargeting protocol in LS174T-bearing mice. Then we compared the pretargeted 177Lu-labeled tetrazine with 177Lu-labeled CC49. The biodistribution data were used for mouse and human dosimetry calculations. Results: The use of a clearing agent led to a doubling of the tetrazine tumor uptake and a 125-fold improvement of the tumor-to-blood ratio at 3 h after tetrazine injection. Mouse dosimetry suggested that this should allow for an 8-fold higher tumor dose than is possible with nonpretargeted radioimmunotherapy. Also, humans treated with CC49-TCO–pretargeted 177Lu-tetrazine would receive a dose to nontarget tissues 1 to 2 orders of magnitude lower than with directly labeled CC49. Conclusion: The in vivo performance of chemical pretargeting falls within the range of results obtained for the clinically validated pretargeting approaches in mice, with the advantage of potentially allowing for fractionated radiotherapy as a result of a lower likelihood of immunogenicity. These findings demonstrate that biologic pretargeting concepts can be translated to rapid bioorthogonal chemical approaches with retained potential.

SPECT/CT imaging of temperature-sensitive liposomes for MR-image guided drug delivery with high intensity focused ultrasound

The goal of this study was to investigate the blood kinetics and biodistribution of temperature-sensitive liposomes (TSLs) for MR image-guided drug delivery. The co-encapsulated doxorubicin and [Gd(HPDO3A)(H₂O)] as well as the ¹¹¹In-labeled liposomal carrier were quantified in blood and organs of tumor bearing rats. After TSL injection, mild hyperthermia (T=42 °C) was induced in the tumor using high intensity focused ultrasound under MR image-guidance (MR-HIFU). The biodistribution of the radiolabeled TSLs was investigated using SPECT/CT imaging, where the highest uptake of ¹¹¹In-labeled TSLs was observed in the spleen and liver. The MR-HIFU-treated tumors showed 4.4 times higher liposome uptake after 48 h in comparison with controls, while the doxorubicin concentration was increased by a factor of 7.9. These effects of HIFU-treatment are promising for applications in liposomal drug delivery to tumors.

Trans-Cyclooctene Tag with Improved Properties for Tumor Pretargeting with the Diels–Alder Reaction

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.

Abstract 4552: Pretargeted radioimmunoimaging and -therapy in tumor-bearing mice using a bioorthogonal reaction

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 111 In-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 125 I-labeled CC49-TCO and 111 In-labeled tetrazine and assessed by dual isotope biodistribution and SPECT imaging with a nanoSPECT/CT. When 111 In-tetrazine was administered to mice 1 day after CC49-TCO, the chemically-tagged tumors reacted rapidly with 111 In-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 111 In-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.