Deglycosylated CD8-specific immunoPET probe prevents antibody-mediated depletion of CD8+ immune cells

1002 Objectives: The biantennary glycans on the heavy chain of IgG antibodies (mAbs) are required for the recognition of antibodies by Fc-gamma receptors (FcγR). However, the Fc portion is a barrier to the development of imaging probes targeting immune cell surface molecules, not only from the pharmacokinetics (PK) standpoint, but from its potential to deplete the effector cells facilitating tumor clearance. Extensive and costly mAb engineering is necessary to remove this fragment. We rationalize that elimination of the heavy chain glycans can resolve immune cell depletion. We examined this hypothesis by comparing the PK, specificity and depleting properties of a 89Zr-labeled (t1/2 ~ 3.27 d) murine anti-CD8 mAb (α-CD8 wt, clone 2.43) against its deglycosylated construct (α-CD8 degly). While 2.43 is a known CD8 T cell depleting antibody, the goal is to evaluate the changes in antibody depletion and tracer development comparing 89Zr-labeled α-CD8 wt and α-CD8 degly. Methods: Deglycosylation was achieved via enzymatic treatment with peptide: N-glycosidase F (PNGaseF) and analyzed via SDS-PAGE. Both α-CD8 wt and degly mAbs were intraperitoneally administered in Balb/c mice (n=3). CD8+ T cell populations were examined by flow cytometry 48 h post-injection (p.i.). For imaging, both mAbs were conjugated to p-SCN-Bn-Desferrioxamine (DFO) at a 1:10 mAb:DFO ratio and labeled with 89Zr. The radiotracers were intravenously injected with 250 μCi (9.25 MBq, 50 μg) in tumor-naive mice to determine spleen uptake and optimal imaging time with time course imaging scans. PK analysis was evaluated via biodistribution at 48 h p.i. We next evaluated the potential of 89Zr-α-CD8 degly to monitor treatment response without depleting CD8 T cell populations. Tracer uptake was analyzed in syngeneic CT26 colorectal tumor bearing-male Balb/c mice dosed with anti-CTLA-4 (100 μg) and anti-PD-1 (200 μg). Results: The SDS-PAGE following PNGaseF incubation confirmed the complete removal of the glycans from the heavy chain of the antibody. From the flow cytometry analysis, α-CD8 wt depleted CD8+ T cells from 15.30% to 0.02% of total viable peripheral blood leukocytes (PBL) at 500 μg compared to α-CD8 degly (23.33%). Tracer development exhibited sufficient labeling at 5 mCi/mg. Spleen uptake in α-CD8 wt mice was consistent from 24 h to 120 h p.i. (11.74 ± 0.40 %ID/g at 24 h, 10.50 ± 0.74 %ID/g at 48 h, 10.90 ± 3.10 %ID/g at 72 h and 11.13 ±1.97 %ID/g at 120 h). The spleen uptake in the α-CD8 degly mice was lower throughout the 120 h: 8.51 ± 0.27 %ID/g at 24 h, 7.89 ± 0.20 %ID/g at 48 h, 7.42 ± 0.83 %ID/g at 72 h and 8.01 ± 0.11 at 120 h p.i. In the biodistribution of tumor-naive mice, the spleen uptake was significantly decreased in the respective 10× blocking suggesting specific targeting of CD8+ T cells in both the α-CD8 wt (35.45 ± 8.14 %ID/g vs. 8.31 ± 2.32 %ID/g block) and α-CD8 degly (29.79 ± 3.06 %ID/g vs. 4.93 ± 0.83 %ID/g block). The biodistribution of the imaging dose exhibited an uptake of 15.08 ± 0.75 %ID/g for α-CD8 wt and 11.68 ± 1.09 %ID/g for α-CD8 degly. Examination of CD8+ T cells in the treated tumors showed no significant difference in uptake at 48 h p.i. (α-CD8 wt: 6.61 ± 0.87 %ID/g tumor and 8.10 ± 1.06 %ID/g spleen; α-CD8 degly 6.02 ± 0.38 %ID/g tumor and 7.60 ± 0.95 %ID/g spleen). The effects of the tracer on the CD8+ T cell viability will be examined and reported. Conclusions: This study proved that eliminating Fc-glycans without completely removing the Fc region from α-CD8 2.43 prevents depletion of immune cell populations critical for tumor clearance. While this study is specific for CD8+ T cells, eliminating heavy chain glycans can be utilized to explore other target cell populations. Importantly, deglycosylation is a simple and cost-effective means to developing exploratory imaging probes to achieve proof-of-concept without investing in expensive antibody engineering techniques.