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

基本信息

批准号：
10533299
负责人：
GEORGE Georgiou GEORGIOU
金额：
$ 46.16万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
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/10533299
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 Macrophage Malignant Neoplasms Maps Measurement Mediating Molecular Myelogenous Myeloid Cells Natural Killer Cells Outcome Pathogenicity Phagocytosis Phenotype Phosphopeptides Phosphorylated Peptide 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 immunological synapse formation improved infectious disease treatment inhibiting antibody microscopic imaging monocyte nano neutrophil novel pathogen phosphoproteomics receptor receptor binding receptor expression sound

项目摘要

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.

项目概要/摘要 Fc介导的抗体效应功能，主要是抗体依赖性细胞吞噬作用（ADCP）和抗体依赖性细胞毒性（ADCC）已被确定在作用机制中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功能。由细胞毒性白细胞亚群上表达的 Fc 受体 (FcR) 的交联触发，如下与多种抗体调理的靶细胞结合最近的发现强调了骨髓-的关键作用。衍生细胞，通过 ADCP/ADCC 清除调理的致病细胞，表达多个 Fc𝛾R，最相关的是激活受体 Fc𝛾RI、Fc𝛾RIIa 和 Fc𝛾RIIIa 以及因为多价免疫复合物 (IC) 会结合并激活所有表面 Fc𝛾R。不同程度的骨髓源性效应子（取决于 Fc𝛾R 表达、Fc:FcR 亲和力、免疫复合物 ADCP 和 ADCC 过程的大小和动力学取决于所有表面 FcR 不同程度激活的综合结果待检验的中心假设。这是通过结扎引发的髓系效应物对 ADCP 和 ADCC 的定量理解通过利用批量检测和高表型含量的单细胞细胞毒性来检测每个单独的 FcR 与磷酸化蛋白质组数据一起进行测定，以绘制骨髓源性效应器上的特定信号转导事件细胞，对于提供一个关于如何设计 Fc 结构域以实现最佳效应器的健全框架至关重要这项工作将利用我们独特的一组结合的非糖基化工程 Fc 结构域。在具体目标 1 中，我们将详尽地介绍对每种 FcR 类型的绝对选择性和拨入亲和力。定量绘制人类执行的效应器表型（ADCC、ADCP、细胞因子释放、trogocytosis）由结扎触发的巨噬细胞和单核细胞（以及中性粒细胞和 NK 细胞，以确保彻底性）每个 FcR 作为亲和力、IC 靶标大小和抗原密度的函数，在 Sp 2 中，我们将使用新型高值。高通量单细胞细胞毒性测定和芯片上细胞计数，以确定免疫的精确动力学 ADCC 中的突触形成和细胞杀伤（或 ADCP 的吞噬）作为 FcR 表达水平的函数单个细胞并询问这些过程的关键相关机制方面。将使用磷酸蛋白质组学来： (a) 识别和定量每个 FcR 触发的肽磷酸化事件 (b) 检测与效应子功能相关的独特 FcR 连接诱导的磷酸肽特征由该受体触发。