Understanding and predicting the consequences of aggregation for the immunogenicity of therapeutic protein products

项目描述：（最多4,000字）请确保此描述清楚地表明该项目如何属于BBSRC的职责范围，它如何实现新的工作方式，以及它如何与DTP主题（世界一流的基础生物科学，工业生物技术和生物能源或农业和粮食安全）保持一致。如果您已经获得了活体技能补充，请概述学生将在项目中学习的活体技能。反复给药治疗性蛋白可导致不必要的免疫原性，以抗药物抗体（ADA）的形式出现，这可能影响药代动力学（PK）、疗效和安全性。高亲和力ADA反应被认为是最有可能影响疗效的，被认为是由B细胞的T细胞依赖性激活触发的。T细胞和B细胞的激活是由治疗蛋白内非自我序列的短工程区域驱动的。T细胞的激活需要抗原呈递细胞（APCs），其内化治疗蛋白，加工并呈递到T细胞表面。单独的抗原呈递不足以激活T细胞，此外还需要共刺激信号，以APC上上调的表面刺激标记的形式，以及来自APC的可溶性细胞因子信号。因此，为了使治疗性蛋白启动ADA反应，apc必须被激活以产生这些共刺激信号。治疗性蛋白质药物治疗的多个方面已被确定为不必要的免疫原性的危险因素，并且有几个方面被提出促进apc的激活，从而向T细胞提供这些共刺激信号。其中一个危险因素是蛋白质聚集。蛋白质聚集体可以在治疗性蛋白质的制造和储存过程中形成，其大小和溶解度范围不同，从由二聚体、三聚体组成的小的、可溶的、可逆的聚集体，到含有大量分子的高阶的、不可逆的聚集体，并且在100 nm - 50 um的亚可见尺寸范围内。对含有低水平这些不同类型聚集体的治疗性蛋白质制剂的研究已被证明在体外引起apc的弱激活，这表明它们在体内可以作为佐剂，并帮助启动对治疗性蛋白质的适应性免疫反应。与BBSRC DTP主题的相关性-生物制剂的免疫原性是开发新的治疗蛋白的主要问题。因此，该项目与工业生物技术主题有关。目的：比较APCs对单体治疗蛋白和不同大小的治疗蛋白的反应。Plan1。选定治疗蛋白的聚合制剂的生成和表征，用作工具试剂（曼彻斯特和剑桥）a)检查聚合条件b)进一步的表征将通过SEC MALLS和其他适当的方法进行。这可能涉及剑桥和曼彻斯特的一些工作。c)用荧光团标记抗体2。在确定的细胞系中摄取和激活的研究-本节的目的是量化聚集蛋白进入apc的摄取量和动力学，并区分其与非聚集物质的行为。我们还将寻求获得一些关于不同摄取途径的信息。使用的方法将是流式细胞仪和共聚焦显微镜。3. 转录组学分析-一旦我们有了一套明确的实验条件，包括特定的细胞系组合，测试含有聚集体的分子和确定的刺激时间点，我们将进行转录组学实验，以确定基因在暴露于聚集体蛋白质和单体时的上调或下调。转化到原代细胞-使用RT-PCR面板，结合显微镜，我们将比较原代细胞中聚集和非聚集物质的摄取

Project Description: (maximum of 4,000 characters)Please make sure this description clearly indicates how the project sits within the BBSRC remit, how it enables new ways of working and how it aligns with the DTP themes (World Class Underpinning Biosciences, Industrial Biotechnology and Bioenergy or Agriculture and Food Security). If you have been awarded an in vivo skills supplement, please outline the in vivo skills the student will learn during the project. Repeated administration of therapeutic proteins can result in unwanted immunogenicity, in the form of anti-drug antibodies (ADA), which can impact on pharmacokinetics (PK), efficacy and safety. High affinity ADA responses, which are considered to be most likely to impact efficacy are believed to be triggered by T cell-dependent activation of B cells. Activation of T and B cells is driven by short engineered regions of non-self-sequences within the therapeutic protein. Activation of T cells requires antigen presenting cells (APCs), which internalise the therapeutic protein, process and present on their surface to T cells. Antigen presentation alone is insufficient for T cell activation, co-stimulatory signals are required in addition, in the form of upregulated surface stimulatory markers on the APC, and soluble cytokine signals, also coming from the APCs. Thus, in order for a therapeutic protein to initiate an ADA response, APCs must be activated to produce these co-stimulatory signals. There are multiple aspects of therapeutic protein drug treatment that have been identified as risk factors for unwanted immunogenicity, and several are proposed to promote activation of APCs, and thus provide these co-stimulatory signals to T cells. One of these risk factors is protein aggregates.Protein aggregates can form during the manufacture and storage of therapeutic proteins, and range in size and solubility, from small, soluble, reversible aggregates consisting of dimers, trimers, to higher-order, irreversible aggregates that contain large numbers of molecules, and fall in the sub-visible size range of 100 nm - 50 um. Studies of therapeutic protein preparations containing low levels of these different types of aggregates have been demonstrated to cause weak activation of APCs in vitro, suggesting that they could function as adjuvants in vivo, and help initiate an adaptive immune response to a therapeutic protein.Relevance to BBSRC DTP Themes - Immunogenicity of biologics is a major problem in the development of new therapeutic proteins. The project therefore relates to the Industrial Biotechnology theme.Aims - To compare the response of APCs to preparations of therapeutic proteins of monomer versus those containing aggregates of different sizes. Plan1. Generation and characterization of aggregated preparations of selected therapeutic proteins to use as tool reagents (Manchester & Cambridge)a) Check aggregation conditionsb) Further characterization will be by SEC MALLS and other methods, as appropriate. This may involve some work in Cambridge, as well as Manchester.c) Label antibodies with fluorophore2. Studies of uptake and activation in defined cell lines - The aim of this section is to quantify uptake quantity and kinetics of aggregated protein into APCs and differentiate behaviour from non-aggregated material. We will also seek to obtain some information about the different uptake pathways used. Methods used will be FACS and confocal microscopy. 3. Transcriptomic analysis - Once we have a well-defined set of experimental conditions with a particular combination of cell line, test molecules containing aggregates and defined stimulation time points, we will conduct a transcriptomic experiment, to identify genes up- or down-regulated in response to exposure to aggregated protein vs monomer4. Translation to primary cells - Using the RT-PCR panel, combined with microscopy, we will compare uptake of aggregated and non-aggregated material in primary cells