Introduction:

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive form of cancer-related malignancies, resulting in the fourth most frequent cause of cancer-related mortalities with a 5-year overall survival of less than 8%.¹ Carbohydrate antigen 19.9 (CA19.9) has been identified as an upregulated cell surface antigen with high surface density in pancreatic cancers, making it an excellent target for antibody imaging and therapeutic applications. Utilizing a pretargeting strategy based on the biorthogonal Diels-Alder click chemistry between trans-cyclooctene (TCO) and tetrazine, is it possible to harness the high specificity of monoclonal antibodies without the pharmacokinetic limitations.² Pretargeting consists of two phases: (1) the injection of a bivalent monoclonal antibody binding to the target antigen but slowly clearing from the blood pool and (2) the injection of the small molecule radioligand able to click the pretargeted tumor cells but with a significantly short plasma half-life.² Such a strategy can mitigate off-site radiotoxicity and enable the use of radionuclides with short half-lives that are usually incompatible with antibodies. Copper-64 (⁶⁴Cu), a positron emitting nuclide and its theranostic match, Copper-67 (⁶⁷Cu), a beta emitting nuclide, are a perfect match for theranostic pretargeting as they exhibit suitable physical half-lives to identify and treat cancer malignancies. In this work, we report on the synthesis, radiolabeling of a novel pretargeting agent, [^64/67Cu]Cu-DiAmSar-Tz₂ and investigate its potential to target PDAC through comparison with a previously studied pretargetting agent, [^64/67Cu]Cu-DiAmSar-Tz (Figure 1A).

Methods and Results:

TCO-NHS was conjugated to 5B1 antibodies via non-site-specific conjugation to terminal lysine residues and injected into nude mice bearing PDAC subcutaneous BxPC-3 xenografts. Two tetrazine precursors, DiAmSar-Tz and DiAmSar-Tz₂, were radiolabeled with ⁶⁴Cu and/or ⁶⁷Cu with radiochemical purity of (>98%). 72 h post 5B1-TCO injection, [^64/67Cu]Cu-DiAmSar-Tz₂ and [^64/67Cu]Cu-DiAmSar-Tz were administered, and their pharmacokinetics were compared through PET-CT imaging and biodistribution studies (1, 4, 24, 48 h). Plasma retention studies confirmed that [⁶⁷Cu]Cu-DiAmSar-Tz₂ cleared slower from the blood up to 30 minutes but cleared similarly to [⁶⁷Cu]Cu-DiAmSar-Tz at later time points. Furthermore, ImmunoPET/CT in vivo images depicted superior localization and adherence of the small molecule at the tumor site at all time points. This was confirmed through a biodistribution study at 24 h and 48 h, demonstrating 6.2 %ID/g for [⁶⁴Cu]Cu-DiAmSar-Tz2 and 1.9 and 1.2 %ID/g for [⁶⁴Cu]Cu-DiAmSar-Tz respectively.

Conclusions:

Compared to its previously investigated monomeric counterpart, the immediate and steady tumor uptake of [^64/67Cu]Cu-DiAmSar-T shows successful vector targeting with minimal off-target accumulation.

Image/Figure:

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Image/Figure Caption:

1:  A) Structure of DiAmSar-Tz and B) DiAmSar-T z 2 . C) 24h PET images of mice bearing PDAC BxPC-3 xenografts injected with 5B1-TCO and [⁶⁴Cu]Cu-DiAmSar-T z 2  or [⁶⁴Cu]Cu-DiAmSar-Tz respectively, confirming increased tumor uptake. D). Ex vivo biodistribution study comparing accumulation of [⁶⁷Cu]Cu-DiAmSar-Tz2 or [⁶⁷Cu]Cu-DiAmSar-Tz at 24 h.

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