Development of Human Asparaginase for Cancer Therapy

描述(由申请人提供)： 这项拟议研究的目标是开发一种使用人类酶而不是细菌酶来治疗血癌的改进方法。目前，这类癌症的主要治疗方法包括注射一种名为天冬酰胺酶的细菌酶。该酶的活性是将天冬酰胺水解为天冬氨酸和氨水。某些血癌依赖于天冬酰胺的胞外池。给予天冬酰胺酶会耗尽这一来源，剥夺癌细胞一种重要的氨基酸，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来自细菌，相当一部分患者对这种酶有免疫反应--这种反应可能是致命的。此外，产生的抗细菌天冬酰胺酶抗体将该酶从循环中清除，从而消除其抗癌潜力，并防止进一步给药。另一个并发症是由于细菌天冬酰胺酶不受欢迎的谷氨酰胺酶活性，这是这种治疗的毒性来源之一。我们建议用人的天冬酰胺酶取代细菌的酶。这将取消免疫反应。我们还将使人类的酶失去谷氨酰胺酶的活性，从而消除这种毒性原因。野生型人天冬酰胺酶的Km值在毫摩尔范围内，不适合替代细菌酶，而血液中天冬酰胺的浓度仅为50微摩尔左右。事实上，临床上使用的来自大肠杆菌和欧文氏菌的细菌酶对天冬酰胺的Km值很低。这项提案中的研究勾勒出了一种策略，使人的天冬酰胺酶具有这种含有天冬酰胺的低Km的重要特性。在目标1中，我们将研究两种人天冬酰胺酶的结构/功能性质。我们将把结构、生化、动力学和诱变方法纳入提案的这一部分。来自目标1的数据将为目标2的工程研究提供信息。策略是引入导致(I)天冬酰胺Km值降低到微摩尔范围的突变，(Ii)失去谷氨酰胺酶活性的酶，以及(Iii)改善热稳定性。后者是为了增加酶在患者体内的循环半衰期，从而使天冬酰胺酶活性持久。目标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。由于人血清白蛋白的循环半衰期为~20天，我们推测融合的人血清白蛋白-天冬酰胺酶相对于游离酶将延长循环半衰期。这将大大有助于该药物的临床使用，并将导致更持久的天冬酰胺耗竭。目的3将测试基因工程天冬酰胺酶在细胞培养中的杀伤力，以及这些酶在血浆中的稳定性。目标3的目的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，接受细菌天冬酰胺酶治疗的成年人表现出更强烈的免疫反应。因此，这里开发的免疫原性较低的酶将特别有利于这一患者群体。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.