posted on 2020-05-12, 16:13authored byJordan
M. White, Outi M. Keinänen, Brendon E. Cook, Brian M. Zeglis, Heather M. Gibson, Nerissa T. Viola
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-CD8wt, clone 2.43), a well-known
depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the
peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly 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-CD8wt was confirmed via SDS-PAGE.
A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue
distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly 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-CD8wt, whereas an increase in CD8+ T cells was shown
with DFO-anti-CD8degly. 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-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. 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.