抗體糖基工程對巨噬細胞介導的抗體依賴性細胞吞噬作用的影響

Abstract

治療性單株抗體（Monoclonal Antibodies, mAbs）被設計用來特異性定位並中和抗原，如癌細胞表達的抗原。通過其片段抗原結合（Fragment Antigen-Binding, Fab）域，mAbs可以誘導癌細胞凋亡或阻斷抑制性信號。此外，mAbs還能通過其片段結晶化（Fc）域觸發其他效應功能。 其中一種效應功能是抗體依賴性細胞吞噬作用（Antibody-Dependent Cellular Phagocytosis, ADCP），由抗體的Fc區域促進。ADCP在具有CD32受體的巨噬細胞中發生。當CD32受體與連接到靶細胞的治療性mAb的Fc區域結合時，巨噬細胞會吞噬並消化mAb結合的細胞。這一過程取決於CD受體與Fc區域的結合能力，而這種結合能力受Fc區域上糖基的影響。糖基工程通過改變這些糖基來增強結合強度，並改善在靶細胞中誘導的效應功能。 Rituximab 被設計為在其 Asn297 糖基化位點具有均一的雙分支 α2,6-唾液酸化複合型糖鏈（α2,6-SCT）、均一的末端半乳糖糖鏈或均一的單葡糖胺（mono-GlcNAc）。我們建立了一種以巨噬細胞為基礎的檢測方法，使用流式細胞術和影像分析來篩選對於抗體依賴性吞噬作用（ADCP）有益的糖型。流式細胞術檢測顯示，α2,6-SCT Rituximab 或末端半乳糖 Rituximab 均能增加 ADCP 活性，相較於原生型 Rituximab。然而，無論是糖工程化的 Rituximab 還是原生型 Rituximab，其 ADCP 活性均高於去糖基化的單葡糖胺（mono-GlcNAc）Rituximab。影像分析檢測同樣顯示，糖基化的抗體（包括糖工程化的或原生型 Rituximab）比單葡糖胺 Rituximab 有更高的 ADCP 活性。這表明糖基化確實在 ADCP 中起作用，但需要進一步研究以確定哪種糖型能誘導最高的 ADCP 活性。

Therapeutic mAbs are designed to specifically locate and neutralize antigens, such as those expressed by cancer cells. Through their fragment antigen-binding (Fab) domain, mAbs can induce apoptosis or block inhibitory signaling in cancer cells. Additionally, mAbs can trigger other effector functions through their fragment crystallizable (Fc) domain. One such effector function is Antibody-Dependent Cellular Phagocytosis (ADCP), facilitated by the Fc region of an antibody. ADCP occurs in macrophages, which have CD32 receptors. When the CD32 receptor binds to the Fc region of a therapeutic mAb attached to a target cell, the macrophage engulfs and digests the mAb-bound cell. This process depends on the ability of the CD receptors to bind to the Fc region, which is influenced by the glycans attached to the Fc region. Glycoengineering modifies these glycans to enhance binding strength and improve the effector functions induced in target cells. Rituximab was engineered to have homogeneous bi-antennary α2,6-sialyl complex-type (α2,6-SCT) glycan, homogeneous terminal galactose glycan, or homogeneous mono-GlcNAc on their Asn297 glycosylation site. We established a macrophage-based assay to screen for glycoforms that are beneficial for ADCP, using both flow cytometry and imaging-based analysis. The flow cytometry-based assay showed that either α2,6-SCT Rituximab or terminal galactose Rituximab increase ADCP activity compared to native Rituximab. However, both glycoengineered Rituximab and native Rituximab has higher ADCP activity compared to deglycosylated mono-GlcNAc Rituximab. The imaging-based assay also showed that glycosylated antibody, both glycoengineered or native Rituximab, has increase ADCP activity compared to mono-GlcNAc Rituximab. This shows that glycosylation does play a role in ADCP, although further study needs to be conducted in order to understand which glycoform is the most effective in elucidating the highest ADCP activity.