The Impact of FcγRI Binding on Immuno-PET

Abstract

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.

Tracers for non-invasive radionuclide imaging of immune checkpoint expression in cancer

“…This can cause anti-target immune activation, complement activation or the formation of anti-drug antibodies, which could lead to target cell depletion, altered tracer pharmacokinetics and serious adverse events when there is cross-recognition of normal proteins; also it limits repetitive imaging in animals. To prevent this, the glycosylation in the Fc domain can be modified, resulting in reduced FcRy mediated uptake (Vivier et al 2019). Despite these drawbacks, many therapeutic immune checkpoint targeting agents are antibodies and by radiolabeling these agents, they can be used for theranostic purposes.…”

“…For example, LAG-3 and TIM-3 play a major role in the activation, proliferation and homeostasis of T cells. Multiple clinical trials are currently ongoing to evaluate the safety and efficacy of pharmaceuticals targeting LAG-3 and TIM-3 and a preclinical study is investigating potential of an imaging tracer directed against LAG-3 (Vivier et al 2019). Another target of interest is V-domain Ig suppressor of T cell activation (VISTA).…”

Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1)

“…The radiopharmaceutical off-target uptake was predominantly abdominal, with the liver and the spleen showing the highest uptake values. This biodistribution pattern is not due to specific TEM1 expression by abdominal organs [5,32], but it is common to all antibodies bearing a functional Fc region [47]. We observed some differences of uptake profiles in the liver and the spleen between the two xenograft tumor models.…”

Development of Antibody Immuno-PET/SPECT Radiopharmaceuticals for Imaging of Oncological Disorders—An Update

“…In immuno-PET/SPECT binding of the Fc region with Fc receptors for example in the reticuloendothelial system, most notably FcγR can result in off-target sequestration and decrease in quality and diagnostic utility of the images. In a recent immuno-PET study tackling this nonspecific uptake, Ab deglycosylation resulted in higher tumor-to-healthy organ contrast and higher quality PET images [ 14 ]. A lower affinity for binding to the FcγR was proposed as mechanism, resulting from the modified 3D configuration of the Fc region [ 15 ].…”