Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with 89 Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding 89 Zrlabeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by ∼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.
Radiolabeled antibodies have shown promise as tools for both the nuclear imaging and endoradiotherapy of cancer, but the protracted circulation time of radioimmunoconjugates can lead to high radiation doses to healthy tissues. To circumvent this issue, we have developed an approach to positron emission tomography (PET) imaging and radioimmunotherapy (RIT) predicated on radiolabeling the antibody after it has reached its target within the body. This in vivo pretargeting strategy is based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). Pretargeted PET imaging and radioimmunotherapy using TCO-modified antibodies in conjunction with Tz-bearing radioligands produce high activity concentrations in target tissues as well as reduced radiation doses to healthy organs compared to directly-labeled radioimmunoconjugates. Herein, we describe how to prepare a TCO-modified antibody (huA33-TCO) as well as how to synthesize two Tz-bearing radioligands: one labeled with the positron-emitting radiometal copper-64 ([ 64 Cu]Cu-SarAr-Tz) and one labeled with the β-emitting radiolanthanide lutetium-177 ([ 177 Lu]Lu-DOTA-PEG7-Tz). We also provide a detailed description of pretargeted PET and pretargeted radioimmunotherapy (PRIT) experiments in a murine model of human colorectal carcinoma. Proper training in both radiation safety and the handling of laboratory mice is required for the successful execution of this protocol.
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