Removal of Fc Glycans from [⁸⁹Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8⁺ T Cells

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of ⁸⁹Zr-radiolabeled CD8-specific murine mAb (anti-CD8_wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8_degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8_wt and anti-CD8_degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with ⁸⁹Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8⁺ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8⁺ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8_wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8_degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [⁸⁹Zr]Zr-DFO-anti-CD8_degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8⁺ T cells in spleens of mice administered with DFO-anti-CD8_wt, whereas an increase in CD8⁺ T cells was shown with DFO-anti-CD8_degly. No statistically significant difference in tumor infiltrating CD8⁺ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [⁸⁹Zr]Zr-DFO-anti-CD8_degly compared to mice imaged with [⁸⁹Zr]Zr-DFO-anti-CD8_wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8⁺ immune molecules with no consequential depletion of their target cell population in peripheral tissues.