The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical halsives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels–Alder click reaction between tetrazine and transcyclooctene. Methods A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen–expressing, SW1222 colorectal cancer xenografts. Results The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA–labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9–10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA–labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either 64Cu or 89Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pre-targeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively). Conclusion The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.

Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron emission tomography (PET) imaging with 18F-fluorodeoxyglucose (18F-FDG). However, 18F-FDG is ineffective in evaluating gliomas due to high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analogue 4-18F-(2S,4R)-fluoroglutamine (18F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced 18F-FGln-tumor avidity, corresponding with decreased tumor burden. 18F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where 18F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that 18F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.

Tumor hypoxia is commonly observed in primary solid malignancies but the hypoxic status of subclinical micrometastatic disease is largely unknown. The distribution of hypoxia in microscopic tumors was studied in animal models of disseminated peritoneal disease and intradermal (i.d.) growing tumors. Tumors derived from human colorectal adenocarcinoma cell lines HT29 and HCT-8 ranged in size from a few hundred microns to several millimeters in diameter. Hypoxia was detected by immunofluorescent visualization of pimonidazole and the hypoxia-regulated protein carbonic anhydrase 9. Tumor blood perfusion, cellular proliferation, and vascularity were visualized using Hoechst 33342, bromodeoxyuridine, and CD31 staining, respectively. In general, tumors of <1 mm diameter were intensely hypoxic, poorly perfused, and possessed little to no vasculature. Larger tumors (f1-4 mm diameter) were well perfused with widespread vasculature and were not significantly hypoxic. Patterns of hypoxia in disseminated peritoneal tumors and i.d. tumors were similar. Levels of hypoxia in microscopic peritoneal tumors were reduced by carbogen breathing. Peritoneal and i.d. tumor models are suitable for studying hypoxia in microscopic tumors. If the patterns of tumor hypoxia in human patients are similar to those observed in these animal experiments, then the efficacy of systemic treatments of micrometastatic disease may be compromised by hypoxic resistance. [Cancer Res 2007;67(16):7646-53]

Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels–Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG7-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with 64Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although 64Cu-Tz-PEG7-NOTA is cleared via both the gastrointestinal and urinary tracts, 64Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, 64Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which 64Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant increases in tumor-to-blood activity concentration ratios. This new strategy offers dosimetric benefits as well, yielding a total effective dose of 0.041 rem/mCi, far below the doses produced by directly labeled 64Cu-NOTA-huA33 (0.133 rem/mCi) and 89Zr-DFO-huA33 (1.54 rem/mCi). Ultimately, this pretargeted PET imaging strategy boasts a dramatically improved pharmacokinetic profile compared to our first generation system and is capable of clearly delineating tumor tissue with high image contrast at only a fraction of the radiation dose created by directly labeled radioimmunoconjugates.

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