Molecular imaging of cell-based therapeutics using an engineered human enzyme.

描述(由申请人提供)：基于细胞的疗法有望给医学带来革命性的变化，适用于多种疾病状态。由于细胞医学的复杂性，它从概念验证到临床应用的过渡一直很缓慢。限制细胞疗法转化的一个主要因素是难以追踪细胞在体内的命运。正电子发射断层扫描(PET)是一种成像技术，它允许以实时和非侵入性的方式进行活体监测。在PET中测量的信号来自放射性同位素，如经历正电子发射的氟-18。因此，含有这样一种同位素的分子--称为PET探针--专门聚集在治疗细胞内，将揭示它们的位置、数量、半衰期等。限制治疗细胞进入临床的另一个因素是对这种新方法不可预见的副作用的担忧。一种允许消除细胞的安全机制将极大地减轻这种担忧。这项提议的长期目标是开发一种双重用途的系统，其中一种是使用PET实时跟踪治疗细胞，另一种是在需要时消除细胞。当探针在治疗细胞中被磷酸化时，PET探针在治疗细胞内的特异性积累就实现了。由于带电分子不能穿过细胞膜，磷酸化使分子被困在细胞内。我们建议将人核苷激酶的基因插入到治疗细胞中，要求该激酶将赋予表达它的细胞独特的活性。PET报告酶的这种独特的酶活性将允许在治疗细胞中优先磷酸化新的PET探针。该领域目前依赖于非人类酶，如HSV1-TK，来实现这种独特的酶活性。然而，作为PET报告酶HSV1-TK有几个缺点，首先是免疫原性。为了避免这一点，我们将使用人胸苷激酶2(TK2)的改良版本作为独特酶活性的来源。为了将这种酶与内源性TK2区分开来，我们将进行酶工程，为我们提供TK2变体，其活性谱与野生型TK2不同，但仍不能引发免疫反应。这项建议的直接目标是通过解决TK2与底物的复合体中的激酶的晶体结构来研究TK2底物专一性的决定因素(目标1)，并利用这一理解来设计对新型PET探针具有独特活性的TK2变体(目标2)。我们将在小鼠模型中测试跟踪表达TK2变体的细胞的能力，并使用HSV1-TK将TK2-系统与现场标准进行比较。这项研究的结果是一种非免疫原性的PET报告酶，具有优化的PET探针活性，将允许在体内实时跟踪治疗细胞。这将对治疗细胞方法向临床的过渡产生重大影响。

DESCRIPTION (provided by applicant): Cell-based therapeutics hold promise to revolutionize medicine, being applicable to numerous disease states. Due to the complex nature of cell-based medicine, its transition from proof-of-concept to clinical use has been slow. A major factor limiting the translation of cell-based therapeutics is the difficulty in tracking the fate of the cells in vivo. Positron emission tomography (PET) is an imaging technology that allows in vivo monitoring in real time and in a non-invasive manner. The signal measured in PET comes from radioactive isotopes, such a fluorine-18, that undergoes positron emission. Thus, a molecule containing such an isotope - called a PET probe - that specifically accumulates inside the therapeutic cells would reveal their location, number, half-life, etc. An additional factor limiting the advancement of therapeutic cells to the clinic has been the concern of unforeseen side effects from this novel approach. A safety mechanism that would allow for the elimination of the cells would greatly diminish this concern. The long-term goal of this proposal is to develop a dual-purpose system that would allow, one, for the real-time tracking of therapeutic cells using PET, and two, for the elimination of the cells if needed. Specific accumulation of the PET probe inside the therapeutic cells is achieved when the probe is phosphorylated in such cells. Phosphorylation traps the molecule within the cell due since charged molecules cannot traverse cell membranes. We propose to insert the gene of a human nucleoside kinase to the therapeutic cells, with the requirement that the kinase would impart unique activity to cells that express it. Such a unique enzymatic activity of a PET reporter enzyme would allow to preferentially phosphorylate novel PET probes in the therapeutic cells. The field currently relies on non-human enzymes, such as HSV1-TK, for that unique enzymatic activity. However, as a PET reporter enzyme HSV1-TK has several drawbacks, foremost being immunogenic. To circumvent this, we will employ a modified version of human thymidine kinase 2 (TK2) as the source for the unique enzymatic activity. To differentiate this enzyme from endogenous TK2, we will perform enzyme engineering to supply us with TK2 variants with an activity profile different to that of wild type TK2, but that still do not elicit an immune response. The immediate goals of this proposal are to study the determinants of substrate specificity of TK2 by solving crystal structures of the kinase in complex with substrates (Aim 1), and to exploit this understanding for the design of TK2 variants with unique activity towards novel PET probes (Aim 2). We will test the ability to track cells expressing our TK2 variants in mouse models, and compare the TK2-system to the standard in the field using HSV1-TK. The results of this research, a non-immunogenic PET reporter enzyme with optimized activity with PET probes, will allow the in vivo real time tracking of therapeutic cells. This would have a dramatic impact on the transition of therapeutic cell approaches to the clinic.