Small molecules to manipulate peptide binding to MHC class I molecules, an optimized method for the generation of MHC tetramers by peptide exchange

MHC（主要组织相容性复合体）I类分子通过在细胞表面将病毒肽呈递给细胞毒性T细胞，在抗病毒免疫反应中发挥关键作用。每个T细胞识别一个特定的i类肽复合物，随后诱导受感染细胞的凋亡。这些特异性T细胞在抗病毒免疫反应过程中增殖，因此检查患者的T细胞库可以鉴定出最具抗原性的肽，从而有可能预测疾病的进展或确定适用于免疫的肽。到目前为止，相关的T细胞是用荧光标记的I类四聚体检测的：四个I类分子与感兴趣的测试肽折叠，生物素化，I类肽复合物通过荧光标记的链亲和素四聚体化。一类四聚体可以有效地与特定的T细胞结合，然后可以检测和量化。一种特殊类型的I类四聚体是所谓的Streptamers：在这里，I类分子携带一个strep标签，它以高亲和力与诱变的链亲和素（StrepTactin）结合，并且可以通过添加生物素从StrepTactin中释放出来，从而实现可逆的T细胞染色。然而，用Streptamers对T细胞进行染色是一项复杂而昂贵的技术，阻碍了免疫学研究和医学治疗。该转移项目的目的是通过改进的Streptamer技术优化T细胞检测，该技术基于我们在dfg资助的项目范围内开发的创新。为了检测特定于不同i类肽复合物的T细胞群，每个Streptamer必须用各自的测试肽从头生成，每次必须重复这个过程需要数周的时间。相比之下，我们将预先生产具有折叠肽的主链tamers，可以在二肽的帮助下交换感兴趣的测试肽，从而将特定链tamer的生产时间减少到几个小时。这一创新不仅将加速和降低T细胞检测的成本，而且将在未来使病毒感染的免疫治疗更加有效。我们的合作伙伴iba GmbH是一家领先的生命科学产品和服务供应商，总部位于德国Göttingen，并以其链球菌标签技术而闻名。在这个转移项目的过程中，我们和iba将共同开发一个原型试剂盒，未来将允许每个诊断和研究实验室单独生成具有感兴趣肽的Streptamers，以节省时间和成本的方式。Iba GmbH随后将出售这些试剂盒，或者提供基于相同检测的T细胞筛查。从长远来看，该项目还将加强德国iba公司在国际竞争对手，特别是美国公司中的市场地位。

MHC (Major Histocompatibility Compex) class I molecules play a pivotal role in the antiviral immune response by presenting viral peptides to cytotoxic T cells at the cell surface. Each T cell recognizes a specific class I-peptide complex and subsequently induces apoptosis of the infected cell. These specific T cells proliferate in the course of an antiviral immune response so that the examination of a patient´s T cell pool allows the identification of the most antigenic peptides offering the possibility to predict the progress of the disease or to determine peptides applicable for immunization.So far, relevant T cells are detected with fluorescently labeled class I tetramers: Four class I molecules are folded with the test peptide of interest, biotinylated, and the class I-peptide complexes are tetramerised via fluorescently labeled streptavidin. A class I tetramer efficiently binds to its specific T cell, which can then be detected and quantified. A special type of class I tetramers are the so-called Streptamers: Here, the class I molecules carry a Strep-tag that binds to a mutagenized streptavidin (StrepTactin) with high-affinity and can be released from StrepTactin by the addition of biotin enabling reversible T cell staining.However, T cell staining with Streptamers is a sophisticated and expensive technique impeding immunological research and medical treatment. The aim of this transfer project is the optimization of T cell detection by an improved Streptamer technology based on an innovation we have developed within the scope of a DFG-funded project. In order to detect T cell populations specific for different class I-peptide complexes, each Streptamer must be generated from scratch with the respective test peptides, a procedure that takes weeks every time it has to be repeated. We, in contrast, will pre-produce master Streptamers with folding peptides, which can be exchanged with the help of dipeptides for a test peptide of interest, thereby reducing the production time of a specific Streptamer to a few hours. This innovation will not only accelerate and cheapen T cell detection but will in the future render immune therapy of viral infections more effective.Our cooperation partner, iba GmbH, is a leading supplier of products and services to the life science community based in Göttingen, Germany, and is especially well known for their Strep-tag technology. In the course of this transfer project, we and iba will together develop a prototype kit that will in the future allow each diagnostic and research lab to individually generate Streptamers with peptides of interest in a time- and cost-saving manner. Iba GmbH will later sell these kits or, alternatively, will offer T cell screenings based on that same assay. In the long term, this project will also strengthen the market position of the German company iba against international competitors, especially US american companies.