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

项目描述：（最多4，000个字符）请确保此描述清楚地表明该项目如何在BBSRC职权范围内，如何实现新的工作方式，以及如何与DTP主题（世界级基础生物科学，工业生物技术和生物能源或农业和粮食安全）保持一致。如果您获得了活体技能补充，请概述学生在项目期间将学习的活体技能。治疗性蛋白质的重复给药可导致以抗药抗体（ADA）形式的不需要的免疫原性，其可影响药代动力学（PK）、功效和安全性。认为最可能影响疗效的高亲和力ADA应答被认为是由T细胞依赖性B细胞活化触发的。T和B细胞的活化由治疗性蛋白质内的非自身序列的短工程化区域驱动。T细胞的活化需要抗原呈递细胞（APC），其内化治疗性蛋白质，加工并在其表面上呈递给T细胞。单独的抗原呈递对于T细胞活化是不够的，另外还需要共刺激信号，其形式为APC上上调的表面刺激标志物，以及可溶性细胞因子信号，其也来自APC。因此，为了使治疗性蛋白质启动ADA应答，必须激活APC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鉴定为不需要的免疫原性的风险因素，并且提出了几个方面来促进APC的活化，从而向T细胞提供这些共刺激信号。这些风险因素之一是蛋白质聚集体，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，并且在大小和溶解度上变化，从由二聚体、三聚体组成的小的、可溶的、可逆的聚集体到包含大量分子的高阶的、不可逆的聚集体，并且落入100 nm - 50 μ m的亚可见大小范围内。含有低水平的这些不同类型的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的弱活化，这表明它们可以在体内起佐剂的作用，并有助于启动对治疗性蛋白质的适应性免疫应答。因此，该项目涉及到工业生物技术的主题。目的-比较APC的单体与那些含有不同大小的聚集体的治疗性蛋白质制剂的反应。计划1。产生和表征用作工具试剂的所选治疗性蛋白质的聚集制剂（Manchester &剑桥）a）检查聚集条件b）进一步表征将通过SEC MALLS和其他方法（视情况而定）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。在确定的细胞系中的摄取和活化研究-本节的目的是量化APC中聚集蛋白的摄取量和动力学，并将行为与非聚集材料区分开来。我们还将寻求获得一些关于所使用的不同摄取途径的信息。使用的方法将是FACS和共聚焦显微镜。3.转录组学分析-一旦我们有了一组定义明确的实验条件，包括细胞系、含有聚集体的测试分子和定义的刺激时间点的特定组合，我们将进行转录组学实验，以鉴定响应于暴露于聚集蛋白与单体4而上调或下调的基因。转化为原代细胞-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