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.

项目总结/摘要 Fc介导的抗体效应子功能，主要是抗体依赖性细胞吞噬作用（ADCP）和抗体依赖性细胞吞噬作用（ADCP）。 依赖性细胞毒性（ADCC），已被确定为在药物的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。效应子功能 由细胞毒性白细胞亚群上表达的Fc受体（FcR）交联引发， 与被多种抗体调理的靶细胞结合。最近的研究结果强调了髓样细胞的关键作用- 衍生的细胞，通过ADCP/ADCC对调理的致病细胞的清除。骨髓衍生效应物， 表达多种Fc受体，最相关的是活化受体Fcγ RI、Fcγ RIIa和Fcγ RIIIa，𝛾 抑制性Fcγ RIIb。由于多价免疫复合物（IC）接合并激活了细胞表面的所有表面Fc受体，𝛾 不同程度的骨髓衍生效应物（取决于Fc：FcR表达、Fc：FcR亲和力、免疫复合物 ADCP和ADCC过程的大小和动力学由以下因素决定： 所有表面FcR不同程度活化的综合结果。待检验的中心假设 在此，通过髓系效应物对ADCP和ADCC的定量理解是通过连接 通过利用批量测定和高表型含量的单细胞细胞毒性， 分析与磷酸化蛋白质组学数据一起绘制髓源性效应物上的特异性信号传导事件 细胞，将是必不可少的，为提供一个健全的框架，就如何工程化Fc结构域的最佳效应 功能协调发展的这项工作将利用我们独特的一组无糖基化的工程化Fc结构域， 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，我们将详尽地和 定量地绘制由人进行的效应器表型（ADCC、ADCP、细胞因子释放、胞啃）， 巨噬细胞和单核细胞（以及嗜中性粒细胞和NK细胞）通过结扎触发 作为亲和力、IC靶大小和抗原密度的函数的每种FcR。在Sp. Aim 2中，我们将使用新的高 通过单细胞细胞毒性测定和芯片上细胞术来确定免疫反应的精确动力学。 ADCC中突触形成和细胞杀伤（或ADCP的吞噬）作为FcR表达水平的函数， 个体细胞，并询问这些过程的关键相关机制方面。在Sp. Aim 3中， 将使用磷酸蛋白质组学：（a）鉴定和定量由每个FcR触发的肽磷酸化事件 和（B）检测与效应子功能相关的独特的FcR连接诱导的磷酸肽特征 由该受体触发。