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说明项目如何位于BBSRC的职权范围内，它如何实现新的工作方式，以及它如何与DTP主题(世界级支撑生物科学、工业生物技术和生物能源或农业和粮食安全)保持一致。如果你已经获得了活体技能补充，请概述学生在项目中将学到的活体技能。重复给予治疗性蛋白会导致不必要的免疫原性，表现为抗药物抗体(ADA)，这可能会影响药代动力学(PK)、疗效和安全性。被认为最有可能影响疗效的高亲和力ADA反应被认为是由T细胞依赖的B细胞激活所触发的。T和B细胞的激活是由治疗性蛋白质中非自身序列的短工程区驱动的。T细胞的激活需要抗原提呈细胞(APC)，它内化治疗蛋白，加工并在其表面呈现为T细胞。单靠抗原提呈不足以激活T细胞，此外还需要共刺激信号，如APC上上调的表面刺激标记，以及来自APC的可溶性细胞因子信号。因此，为了使治疗性蛋白质启动ADA反应，APC必须被激活以产生这些共刺激信号。治疗蛋白药物治疗有多个方面被确定为不想要的免疫原性的危险因素，有几个方面被提出以促进APC的激活，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，其大小和溶解度范围从由二聚体、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聚集体，并落在100 nm-50微米的亚可见尺寸范围内。研究表明，含有低水平这些不同类型聚集体的治疗性蛋白制剂在体外会导致APC的微弱激活，这表明它们可以在体内作为佐剂发挥作用，并有助于启动对治疗性蛋白的适应性免疫反应。与BBSRC DTP主题的关系-生物制品的免疫原性是开发新的治疗性蛋白的主要问题。因此，该项目与工业生物技术主题有关。目的-比较APC对单体治疗蛋白制剂和含有不同大小聚集体的APC的反应。计划1。选定的治疗性蛋白质聚合制剂的生成和表征以用作工具试剂(曼彻斯特和剑桥)a)检查聚集条件b)进一步表征将由SEC商场和其他适当的方法进行。这可能涉及剑桥大学的一些工作，以及曼彻斯特用荧光团标记抗体。对特定细胞系的摄取和激活的研究-本节的目的是量化聚集蛋白进入APC的摄取量和动力学，并将行为与非聚集物质区分开来。我们还将寻求获得有关所使用的不同吸收途径的一些信息。使用的方法是流式细胞仪和共聚焦显微镜。3.转录分析-一旦我们有了一组明确的实验条件，包括细胞系、含有聚集体的测试分子和确定的刺激时间点的特定组合，我们将进行转录实验，以识别与单体蛋白接触时上调或下调的基因4。转化为原代细胞-使用RT-PCR板，结合显微镜，我们将比较聚集和非聚集物质在原代细胞中的摄取

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