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

使用工程人类酶对基于细胞的疗法进行分子成像。

基本信息

批准号：
8704931
负责人：
ARNON LAVIE
金额：
$ 34.52万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8704931
关键词：
2&apos -fluoro-5-methylarabinosyluracil Address Adverse effects Affect Animal Model Carbon Cell membrane Cell physiology Cells Cellular Immunity Charge Clinic Clinical Complex Deoxycytidine Deoxyglucose Development Disease Engineered Gene Engineering Enzymes Feedback Fluorine Genes Goals Half-Life Health Herpesvirus 1 Human Human Engineering Image Imagery Imaging technology Immune response Isotopes Life Location Measures Medicine Methods Modification Molecular Monitor Names Nature Normal tissue morphology Nucleosides Patients Phosphorylation Phosphotransferases Physiological Positron Positron-Emission Tomography Radioisotopes Radiopharmaceuticals Regenerative Medicine Reporter Reporter Genes Research Safety Signal Transduction Source Specificity Structure Substrate Specificity System T-Lymphocyte Technology Testing Therapeutic Therapeutic Uses Thymidine Time Tissues Translations Variant base cancer cell catalyst cellular imaging clinical application design enzyme substrate gain of function glucose analog hexokinase human TK2 protein immunogenic immunogenicity improved in vivo induced pluripotent stem cell interest molecular imaging mouse model mutant novel novel strategies nucleoside analog nucleoside kinase tumor

项目摘要

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.

描述（由申请人提供）：基于细胞的疗法有望彻底改变医学，适用于多种疾病状态。由于细胞医学的复杂性，其从概念验证到临床应用的转变一直很缓慢。限制细胞疗法转化的一个主要因素是难以追踪体内细胞的命运。正电子发射断层扫描（PET）是一种能够以非侵入方式实时监测体内的成像技术。 PET 中测量的信号来自放射性同位素，例如氟 18，它会发射正电子。因此，含有这种同位素的分子（称为 PET 探针）可以在治疗细胞内特异性积累，从而揭示其位置、数量、半衰期等。限制治疗细胞向临床发展的另一个因素一直是人们关注的问题。这种新颖方法带来的不可预见的副作用。允许消除细胞的安全机制将大大消除这种担忧。该提案的长期目标是开发一种双用途系统，一是使用 PET 实时跟踪治疗细胞，二是在需要时消除细胞。当探针在治疗细胞中被磷酸化时，PET 探针就会在治疗细胞内特异性积累。由于带电分子无法穿过细胞膜，磷酸化将分子捕获在细胞内。我们建议将人类核苷激酶的基因插入到治疗细胞中，要求该激酶能够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优先磷酸化治疗细胞中的新型 PET 探针。该领域目前依赖于非人类酶，例如 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，最重要的是具有免疫原性。为了避免这种情况，我们将采用改良版的人胸苷激酶 2 (TK2) 作为独特酶活性的来源。为了区分这种酶与内源 TK2，我们将进行酶工程，为我们提供 TK2 变体，其活性特征与野生型 TK2 不同，但仍然不会引起免疫反应。该提案的直接目标是通过解决激酶与底物复合物的晶体结构来研究 TK2 底物特异性的决定因素（目标 1），并利用这种理解来设计对新型 PET 探针具有独特活性的 TK2 变体（目标 2）。我们将测试在小鼠模型中追踪表达 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 PET 报告酶，通过 PET 探针优化其活性，将能够在体内实时跟踪治疗细胞。这将对细胞治疗方法向临床的转变产生巨大影响。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A Novel l-Asparaginase with low l-Glutaminase Coactivity Is Highly Efficacious against Both T- and B-cell Acute Lymphoblastic Leukemias In Vivo.

DOI：
10.1158/0008-5472.can-17-2106
发表时间：
2018-03-15
期刊：
Cancer research
影响因子：
11.2
作者：
Nguyen HA;Su Y;Zhang JY;Antanasijevic A;Caffrey M;Schalk AM;Liu L;Rondelli D;Oh A;Mahmud DL;Bosland MC;Kajdacsy-Balla A;Peirs S;Lammens T;Mondelaers V;De Moerloose B;Goossens S;Schlicht MJ;Kabirov KK;Lyubimov AV;Merrill BJ;Saunthararajah Y;Van Vlierberghe P;Lavie A
通讯作者：
Lavie A

Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.