Systematic, molecular level analysis of the Fc receptor ligation on antibody effector functions

项目摘要/摘要 Fc介导的抗体效应器功能，主要是抗体依赖细胞吞噬(ADCP)和抗体 依赖细胞毒性(ADCC)，已被建立起核心作用的作用机制 治疗性抗体，包括抗感染抗体和免疫检查点抑制剂。效应器函数 是由表达在下列细胞毒性白细胞亚群上的Fc受体(FCR)的交联性触发的 与多种抗体调理的靶细胞结合。最近的发现突显了髓系细胞的关键作用- 来源细胞对ADCP/ADCC清除调理的致病细胞的作用。髓系来源的效应器， 表达多个Fc𝛾受体，最相关的是激活受体Fc𝛾RI、Fc𝛾Ria和Fc𝛾RIIa以及 抑制性Fc𝛾RIIb。因为多价免疫复合体(IC)结合并激活所有表面Fc𝛾受体 不同程度的髓系效应器(取决于Fc𝛾R的表达、Fc：Fcr亲和力、免疫复合体 大小和抗原密度)ADCP和ADCC过程的大小和动力学由 所有表面FCR不同程度激活的综合结果。需要检验的中心假说 这就是髓系效应分子对ADCP和ADCC的定量理解 利用批量检测和高表型含量单细胞细胞毒性的优势，每个个体FCR 结合磷蛋白质组数据分析髓系效应器上的特异性信号事件 细胞，对于提供关于如何设计FC域以获得最佳效应器的合理框架将是必不可少的 功能。这项工作将利用我们独特的一组糖基化工程Fc结构域，这些Fc结构域与 对每种FCR类型的绝对选择性和拨入亲和力。在具体目标1中，我们将穷尽和 定量绘制人类执行的效应表型(ADCC、ADCP、细胞因子释放、巨噬细胞增多症) 由结扎触发的巨噬细胞和单核细胞(以及由中性粒细胞和NK细胞触发) 作为亲和力、IC靶标大小和抗原密度的函数。在Sp.目标2我们将使用新颖的HIGH 单细胞细胞毒试验和芯片上细胞仪测定免疫的精确动力学 ADCC(或ADCP吞噬)中突触的形成和细胞杀伤与FCR表达水平的关系 并审问对这些进程至关重要的关键相关机制方面。在Sp.目标3我们 将使用磷酸蛋白质组学：(A)识别和量化由每个FCR触发的多肽磷酸化事件 以及(B)检测与效应器功能相关的独特的FCR连接诱导的磷酸肽特征 由该受体触发。

PROJECT SUMMARY/ABSTRACT Fc-mediated antibody effector functions, primarily antibody dependent cell phagocytosis (ADCP) and antibody dependent cell cytotoxicity (ADCC), have been established to play a central role on the mechanism of action of therapeutic antibodies including anti-infective antibodies and immune checkpoint inhibitors. Effector functions are triggered by the crosslinking of Fc receptors (FcRs) expressed on cytotoxic leukocyte subsets following binding to target cells opsonized by multiple antibodies. Recent findings have highlighted the key role of myeloid- derived cells, on the clearance of opsonized pathogenic cells by ADCP/ADCC. Myeloid-derived effectors, express multiple Fc𝛾Rs, most relevant being the activating receptors Fc𝛾RI, Fc𝛾RIIa and Fc𝛾RIIIa and the inhibitory Fc𝛾RIIb. Because multivalent immune complexes (ICs) engage and activate all surface Fc𝛾Rs on myeloid-derived effectors to various degrees (depending on Fc𝛾R expression, Fc:FcR affinity, immune complex size and antigen density) the magnitude and kinetics of the ADCP and ADCC processes are determined by the integrated outcome of the activation of all surface FcRs to various degrees. The central hypothesis to be tested here is that the quantitative understanding of ADCP and ADCC by myeloid effectors triggered by the ligation of each individual FcR by taking advantage of bulk assays and high phenotypic content single-cell cytotoxicity assays together with phosphoproteomic data to map the specific signaling events on myeloid-derived effector cells, will be essential for providing a sound framework on how to engineer the Fc domain for optimal effector functions. This work will capitalize on our unique set of aglycosylated engineered Fc domains that bind with absolute selectivity and dialed-in affinity to each FcR type. In Specific Aim 1 we will exhaustively and quantitatively map the effector phenotypes (ADCC, ADCP, cytokine release, trogocytosis) performed by human macrophages and monocytes (as well as by neutrophils and by NK cells for thoroughness) triggered by ligation of each FcR as a function of affinity, IC target size and antigen density. In Sp. Aim 2 we will use novel high throughput single cell cytotoxicity assays and on-chip cytometry to determine the precise kinetics of immune synapse formation and cell killing in ADCC (or engulfment for ADCP) as a function of FcR expression levels on individual cells and to interrogate key relevant mechanistic aspects central to these processes. In Sp. Aim 3 we will use phosphoproteomics to: (a) identify and quantitate peptide phosphorylation events triggered by each FcR and (b) detect unique FcR ligation-induced phosphopeptide signatures that correlate with effector functions triggered by that receptor.