从[89Zr] Zr-DFO-Anti-CD8中除去Fc聚糖可防止CD8 + T细胞的周边消耗。,Molecular Pharmaceutics

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从[89Zr] Zr-DFO-Anti-CD8中除去Fc聚糖可防止CD8 + T细胞的周边消耗。
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2020-04-24 , DOI: 10.1021/acs.molpharmaceut.0c00270
Jordan M White ₁ , Outi M Keinänen _{2,

3} , Brendon E Cook _{2,

4} , Brian M Zeglis _{2,

3,

4} , Heather M Gibson ₁ , Nerissa T Viola ₁

Affiliation

在Ñ -连接上的免疫球蛋白G（IgG）的抗体（mAb）是在通过FC-γ受体（FcγR的）识别的Fc区的仪器重链双触角聚糖。在针对免疫细胞群的全长基于mAb的成像示踪剂的情况下，这些Fc：FcγR相互作用可能会耗尽负责清除肿瘤的效应细胞。为了绕过这个问题，我们假设通过酶法去除Fc聚糖会破坏Fc：FcγR相互作用，并在免疫正电子发射断层扫描（immunoPET）成像过程中消耗掉以示踪剂为靶标的免疫细胞。在本文中，我们比较了在体外和在体内的性质⁸⁹ Zr的放射性标记CD8特异性鼠mAb（抗CD8_重量（克隆2.43），一个著名的消耗单抗及其去糖基化对应物（抗CD8 _degly）。通过用肽N-糖苷酶F（PNGaseF）进行酶处理来实现去糖基化。将抗-CD8 _wt和抗-CD8 _degly mAb都缀合到对-SCN-Bn-去铁胺（DFO），并用⁸⁹ Zr标记。比较了两种DFO偶联的mAb与FcγR和CD8 ⁺脾细胞的结合。进行了体内成像和分布研究，以检查未免疫肿瘤和CT26结直肠肿瘤的小鼠中放射免疫偶联物的特异性和药代动力学。CD8 ^+的离体分析通过流式细胞术和qRT-PCR测量脾脏中的T细胞群体和成像后获得的肿瘤。除去从抗CD8与Fc聚糖的_重量经SDS-PAGE证实。DFO-抗-CD8 _degly表现出FcγR相互作用的降低，而其与CD8的结合保持不变。组织分布显示[ ⁸⁹ Zr] Zr- _DFO-抗CD8 _degly和wt放射免疫_缀合物具有相似的药代动力学。体内阻断研究进一步证明了去糖基化放射性示踪剂对CD8的保留特异性。根据影像学研究，两种放射性示踪剂之间均未观察到脾脏和肿瘤中积累的差异。流式细胞仪分析的结果证实CD8已耗尽施用DFO-抗CD8 _wt的小鼠脾脏中的⁺ T细胞，而_DFO-抗CD8 _degly显示CD8 ⁺ T细胞增加。当与对照未调节小鼠相比时，在与探针一起施用的队列中未观察到肿瘤浸润CD8 ⁺ T细胞的统计学显着差异。从切除的肿瘤CD8 mRNA水平显示增加了抗原的转录物结合的小鼠[ ⁸⁹锆]的Zr-DFO-抗CD8 _degly与用[成像小鼠⁸⁹的Zr]的Zr-DFO-抗CD8_重量。总之，去除Fc聚糖提供了一种直接的方法来开发基于全长抗体的成像探针，专门用于检测CD8 ⁺免疫分子，而不会因此而耗尽周围组织中的靶细胞。

"点击查看英文标题和摘要"

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.

更新日期：2020-04-24

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