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

Abstract

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.

Keywords: CD8 imaging; Fc glycan removal; immune cell depletion; immunoPET.

Figure 1.

Characterization of anti-CD8_degly. (A) PNGaseF deglycosylation of anti-CD8 clone 2.43. Lane 1, MW ladder; 2, anti-CD8_degly; 3, anti-CD8_wt; 4, deglycosylation reaction; 5, PNGaseF; 6, MW ladder. (B) Optical density (OD) values at 450 nm were obtained in quadruplicate by ELISA to compare the Fc-FcγR binding following deglycosylation. In contrast to DFO-anti-CD8_wt, DFO-anti-CD8_degly had diminished binding to mFcγRI. *** p = 0.0004.

Figure 2.

DFO-anti-CD8_degly does not deplete the CD8⁺ T cell population. Flow cytometry analysis of spleens of (A) untreated mice, mice administered with DFO-anti-CD8_wt (B) 50 μg or (C) 500 μg, and mice treated with DFO-anti-CD8_degly (D) 50 μg or (E) 500 μg. In the untreated mice, a CD8⁺ T cell population was observed in Q3. The CD8⁺ T cell population was not present in the anti-CD8_wt mice as exhibited by the decrease in the cell population in Q4. The percentages noted in the quadrants for panels A–E are representative of one mouse. (F) CD8⁺ T cells analysis expressed as mean ± SD from one experiment (n = 3 mice/group) and analyzed by one-way ANOVA with a Tukey’s posthoc analysis. **** p < 0.0001 versus wt. $$ p = 0.0013, $$$ p < 0.001, $$$$ p < 0.0001 versus control.

Figure 3.

In vivo imaging of CD8 in BALB/c male mice. (A) Time activity curve of spleen uptake of both [⁸⁹Zr]Zr-DFO-anti-CD8_wt and [⁸⁹Zr]Zr-DFO-anti-CD8_degly identifies similar tracer uptake from 24 to 120 h p.i. (n = 3 mice/group, n = 5 mice/group at 48 h). (B) Maximum intensity projections (MIP, top panels) and planar sections (bottom panels) demonstrate overall distribution and uptake of both [⁸⁹Zr]Zr-DFO-anti-CD8_wt (left) and [⁸⁹Zr]Zr-DFO-anti-CD8_degly (right) at 48 h p.i. S = spleen. ** p < 0.01.

Figure 4.

Ex vivo validation of tracer specificity. (A) Tissue distribution of the [⁸⁹Zr]Zr-DFO-anti-CD8 tracers (n = 5/tracer for each time point) exhibits nominal uptake in organs with the highest uptake in the spleen (inset). Blocked cohorts (n = 5/tracer) administered with 10-fold excess cold unmodified antibody respectively exhibited decreased tracer uptake in the spleen. (B) To examine mass effects on the pharmacokinetics of the tracers, an imaging dose of 200–250 μCi of tracer was intravenously injected. Splenic uptake remained the highest but displayed lower accumulation in comparison to the biodistribution dose (A). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Stom. = Stomach, Sm. Int. = Small Intestines, Lg. Int. = Large Intestines, Panc. = Pancreas.

Figure 5.

In vivo CT26 tumor imaging and ex vivo flow cytometric analysis. (A) Maximum intensity projections (MIP, top left) and planar sections (bottom right) of [⁸⁹Zr]Zr-DFO-anti-CD8_wt (n = 5) and [⁸⁹Zr]Zr-DFO-anti-CD8_degly (n = 5) in CT26 immunogenic colorectal tumors (T) showed no difference in uptake (right). (B) mRNA analysis of CD8 in the tumor showed a decrease in CD8 transcripts in the [⁸⁹Zr]Zr-DFO-anti-CD8_wt imaged mice compared to the [⁸⁹Zr]Zr-DFO-anti-CD8_degly mice. (C) IFN-γ transcripts were significantly lower in the [⁸⁹Zr]Zr-DFO-anti-CD8_wt injected mice. mRNA studies were completed in triplicate from n = 5 mice/group and n = 2 samples from the CT26 cell line. Statistical analysis of mRNA was conducted by one-way ANOVA with a Tukey’s post hoc analysis. (D) Flow cytometric analysis of dissociated CT26 from mice injected with nonradiolabeled tracers exhibited no difference in CD8⁺ T cells. (E) In spleens, DFO-anti-CD8_wt exhibited lower CD8⁺ T cells compared to control and DFO-anti-CD8_degly; DFO-anti-CD8_degly exhibited significantly higher CD8⁺ T cells compared to control and wt.