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

Fc 受体连接对抗体效应子功能的系统分子水平分析

• 批准号: 10308041
• 负责人: GEORGE Georgiou GEORGIOU
• 金额: $ 46.2万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-19 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308041
• 关键词: Affinity; Anti-Infective Agents; Antibodies; Antigen-Antibody Complex; Antigens; Area; Binding; Biological Assay; Biological Products; Cell Cycle Kinetics; Cell surface; Cells; Coupled; Cytometry; Data; Development; Effector Cell; Engineering; Event; Fc Receptor; Fc domain; Glean; Human; IgG1; Immune checkpoint inhibitor; Individual; Kinetics; Leukocytes; Ligation; Malignant Neoplasms; Maps; Measurement; Mediating; Molecular; Myelogenous; Myeloid Cells; Natural Killer Cells; Outcome; Pathogenicity; Peptides; Phagocytosis; Phenotype; Phosphopeptides; Phosphorylation; Play; Process; Property; Role; Signal Transduction; Surface; Synapses; Testing; Therapeutic Monoclonal Antibodies; Therapeutic antibodies; Work; antibody-dependent cell cytotoxicity; cell killing; clinical development; clinical efficacy; crosslink; cytokine; cytotoxic; cytotoxicity; density; exhaustion; immunological synapse formation; improved; infectious disease treatment; inhibiting antibody; macrophage; microscopic imaging; monocyte; neutrophil; novel; pathogen; phosphoproteomics; receptor; receptor binding; receptor expression; sound

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.

项目摘要/摘要 FC介导的抗体效应子功能，一级抗体依赖性细胞吞噬作用（ADCP）和抗体 依赖性细胞细胞毒性（ADCC）已建立，以在作用机理上发挥核心作用 治疗性抗体，包括抗感染抗体和免疫检查点抑制剂。效应器功能 是由在细胞毒性白细胞子集上表达的FC受体（FCR）的交联引发的 与靶细胞结合多种抗体。最近的发现强调了髓样的关键作用 - 衍生细胞，通过ADCP/ADCC清除致病性病原细胞。髓样衍生的作用， 表达多个FC𝛾RS，最相关的是激活受体FC𝛾RI，FC𝛾RIIA和FC𝛾RIIIA，以及 抑制性fc𝛾riib。因为多价免疫复合物（ICS）在 髓样衍生的效果在各种程度上（取决于FC𝛾R表达，FC：FCR亲和力，免疫复合物 大小和抗原密度）ADCP和ADCC过程的幅度和动力学由 所有表面FCR激活到各个程度的综合结果。要测试的中央假设 这是通过连接触发的对ADCP和ADCC的定量理解 每个单独的FCR通过利用大量测定和高表型含量单细胞毒性 测定与磷酸蛋白质组学数据一起绘制髓样衍生效应子上的特定信号事件 单元格，对于如何为如何设计FC域以提供最佳效应器至关重要 功能。这项工作将利用我们独特的汇总FC域，这些域结合 绝对选择性和对每种FCR类型的拨入亲和力。在特定目标1中，我们将详尽地 定量绘制人类进行的效应表型（ADCC，ADCP，细胞因子释放，trogocytosis） 巨噬细胞和单核细胞（以及中性粒细胞和NK细胞以彻底）触发 每个FCR作为亲和力，IC目标大小和抗原密度的函数。在sp。目标2我们将使用小说高 吞吐量单细胞毒性测定和片上细胞术，以确定免疫的精确动力学 ADCC中的突触形成和细胞杀死（或ADCP吞噬）作为FCR表达水平的函数 单个细胞并询问关键相关的机械方面，这是这些过程中心的核心。在sp。目标3我们 将使用磷酸蛋白质组学来：（a）识别和定量由每个FCR触发的肽磷酸化事件 （b）检测与效应功能相关的独特FCR连接诱导的磷酸肽特征 由该接收器触发。