A first-of-its-kind 18F pretargeted PET imaging approach based on the bioorthogonal inverse electron demand Diels–Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is presented. As proof-of-principle, a TCO-bearing immunoconjugate of the anti-CA19.9 antibody 5B1 and an Al[18F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and 18F-PET imaging data clearly demonstrate that this methodology effectively delineates tumor mass with activity concentrations up to 6.4 %ID/g at 4 h after injection of the radioligand.

HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

5B1 is a fully human, monoclonal antibody that has shown promise for the PET imaging of cancers expressing carbohydrate antigen 19.9 (CA19.9)—a carbohydrate prevalent in cells with aberrant glycosylation and an established effector of metastasis. The long physiologic half-life of the antibody and interference from circulating CA19.9 may increase the time required to generate quality images as well as the risk of radiation exposure to healthy tissues during repeated PET imaging. Pretargeting methodologies are an effective approach to expeditiously acquire PET images, but in this case, the pretargeting approach is complicated by the internalization of 5B1 by CA19.9-expressing cells. We sought to adapt and optimize a pretargeting strategy that exploits the bioorthogonal reaction between transcyclooctene (TCO) and tetrazine (Tz) to overcome these complications. Methods 5B1 was modified with TCO, and a novel NOTA-PEG7-Tz radioligand was synthesized with the goal of improving on a previously reported analog. BxPC3 and Capan-2 cells were evaluated for their ability to internalize anti-CA19.9 antibodies using a fluorometric assay, and xenografts of the same lines were used for in vivo studies. The pretargeting approach was optimized, and the 2 radioligands were compared using biodistribution and PET imaging in murine models of pancreatic cancer. Results BxPC3 and Capan-2 cells were shown to rapidly internalize anti-CA19.9 monoclonal antibodies, including 5B1. 64Cu-NOTA-PEG7-Tz showed improved in vivo pharmacokinetics relative to 64Cu-NOTA-Tz using 5B1-TCO as the targeting vector. PET imaging and biodistribution studies showed that injecting the radioligand 72 h after the administration of 5B1-TCO resulted in the best uptake (8.2 ± 1.7 percentage injected dose per gram at 20 h after injection) and tumor-to-background activity concentration ratios. Dosimetry calculations revealed that the pretargeting system produced a greater than 25-fold reduction in total body radiation exposure relative to 89Zr-desferrioxamine-5B1. PET/CT imaging in an orthotopic Capan-2 xenograft model—which secretes large amounts of CA19.9 and more rapidly internalizes anti-CA19.9 antibodies—showed that this approach is viable even in the difficult circumstances presented by a circulating antigen and internalized targeting vector. Conclusion The 5B1-TCO and 64Cu-NOTA-PEG7-Tz system evaluated in these studies can delineate CA19.9-positive xenografts in murine models of pancreatic cancer despite the challenges posed by the combination of circulating antigen and internalization of the 5B1-TCO.

The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Towards that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1— a human, anti-CA19.9 mAb — in preclinical murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8 %ID/g at 4h) and persistent (16.8 ± 3.9 %ID/g at 120h) uptake in tumors while concurrently clearing from blood and non-target tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of 177Lu-DOTA-PEG7-Tz (400, 800, and 1200µCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e. liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation.