Development of Human Asparaginase for Cancer Therapy

描述（由申请人提供）： 这项研究的目的是开发一种利用人类酶而不是细菌酶治疗血癌的改进方法。目前，这类癌症的主要治疗方法包括施用一种称为天冬酰胺酶的细菌酶。这种酶的活性是将氨基酸天冬酰胺水解为天冬氨酸和氨。某些血癌依赖于天冬酰胺的细胞外库。天冬酰胺酶的施用耗尽了这种来源，剥夺了癌细胞的重要氨基酸，并最终诱导细胞死亡（细胞凋亡）。 由于目前批准的所有天冬酰胺酶制剂都是细菌来源的，因此相当大比例的患者对这种酶产生免疫反应，这种反应可能是致命的。 此外，所产生的抗细菌天冬酰胺酶抗体从循环中清除酶，从而消除其抗癌潜力并防止药物的进一步施用。另一个并发症是由于细菌天冬酰胺酶的不期望的天冬酰胺酶活性，这是该治疗的毒性来源。我们建议用人天冬酰胺酶代替细菌酶。 这将消除免疫反应。我们还将设计人体酶，使其不具有转氨酶活性，从而消除这种毒性。 人天冬酰胺酶的野生型版本不适合替代细菌酶，因为它们的Km值在毫摩尔范围内，而血液中天冬酰胺的浓度仅为约50微摩尔。事实上，临床上使用的细菌酶，来自E。大肠杆菌和欧文氏菌对天冬酰胺的Km值较低。本提案中的研究描绘了一种策略，以工程改造人类天冬酰胺酶，使其具有与天冬酰胺的低Km的重要特性。 在目的1中，我们将研究两种人天冬酰胺酶的结构/功能特性。我们将把结构，生物化学，动力学和诱变的方法在这部分的建议。 目标1的数据将为目标2的工程研究提供信息。该策略是引入突变，其导致（i）天冬酰胺Km值降低至微摩尔范围，（ii）缺乏天冬酰胺酶活性的酶，和（iii）改善的热稳定性。后者是为了增加酶在患者体内的循环半衰期，使天冬酰胺酶活性持久。目的2的另一个新方面是人天冬酰胺酶与人血清白蛋白（HSA）的连接。由于HSA具有约20天的循环半衰期，我们假设融合HSA-天冬酰胺酶相对于游离酶将具有增加的循环半衰期。这将显着有助于该药物的临床使用，并将导致更持久的天冬酰胺消耗。 目的3将测试工程天冬酰胺酶在细胞培养中的细胞杀伤能力，以及酶在血浆中的稳定性。目的3是为临床试验准备工程化的人天冬酰胺酶。 与儿科患者相比，用细菌天冬酰胺酶治疗的成人表现出更强烈的免疫应答。因此，本文开发的免疫原性较低的酶将特别有益于该患者群体。这使得这项建议与退伍军人的待遇特别相关。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop an improved approach for the treatment of blood cancers that utilizes human enzymes rather than bacterial enzymes. Currently, a mainstay treatment of such cancers includes the administration of a bacterial enzyme called asparaginase. The activity of this enzyme is to hydrolyze the amino acid asparagine to aspartic acid and ammonia. Certain blood cancers are dependent on the extracellular pool of asparagine. Administration of asparaginase depletes this source, depriving the cancer cells of a vital amino acid, and ultimately induces cell death (apoptosis). Because all currently approved preparations of asparaginases are of bacterial origin, a substantial proportion of patients have an immune response against the enzyme - a reaction that can be deadly. Moreover, the generated anti-bacterial asparaginase antibodies clear the enzyme from circulation, thereby eliminating its anticancer potential and preventing further administration of the drug. A further complication is due to the undesired glutaminase activity of bacterial asparaginases, which is a source of toxicity of this treatment. We propose to replace the bacterial enzymes with human asparaginases. This will abolish the immune response. We will also engineer the human enzymes to be devoid of glutaminase activity, thereby eliminating this cause of toxicity. The wild-type versions of human asparaginases are not suitable for replacing the bacterial enzymes since their Km value is in the millimolar range, yet the concentration of asparagine in blood is only about 50 micromolar. Indeed, the bacterial enzymes used in the clinic, from E. coli and Erwinia, have a low Km value for asparagine. The research in this proposal delineates a strategy to engineer human asparaginases to have this vital property of low Km with asparagine. In Aim 1 we will study the structure/function properties of two human asparaginases. We will incorporate structural, biochemical, kinetic, and mutagenesis approaches in this part of the proposal. The data from Aim 1 will inform the engineering studies of Aim 2. The strategy is to introduce mutations that result in (i) lowering of the asparagine Km value to the micromolar range, (ii) an enzyme devoid of glutaminase activity, and (iii) improved thermo-stability. The latter is to increase the circulation half-life of the enzyme in the patientsso that the asparaginase activity is long lasting. An additional novel aspect of Aim 2 is the linking f the human asparaginase to human serum albumin (HSA). Since HSA has a circulation half-life of ~20 days, we hypothesize that the fusion HSA- asparaginase will have increased circulation half-life relative to the free enzyme. This will significantly aid the clinical use of this drug, ad will result in more persistent asparagine depletion. Aim 3 will test the cell-killing power of the engineered asparaginases in cell culture, and the stability of the enzymes in plasma. The purpose of Aim 3 is to ready the engineered human asparaginases for clinical trials. Adults treated with the bacterial asparaginases exhibit a more intense immune response compared to pediatric patients. Hence, the less immunogenic enzymes developed here will especially be beneficial to this patient population. This makes this proposal especially relevant to the treatment of veterans.