Pharmacological and toxicological testing of a novel L-asparaginase

项目摘要：本提案的目标是对一种显著更安全的变体进行IND研究 抗癌生物药物左旋门冬酰胺酶的研究。L-天冬酰胺酶是酶药物，其作用是消耗 血液中的天冬酰胺由于毒性，这是特别明显的成人，L- 天冬酰胺酶治疗仅限于急性淋巴细胞白血病（ALL），一种白色血细胞的癌症。一 L-天冬酰胺酶的毒性来源是由于它们的细菌来源（来自E.大肠杆菌（Elspar）或欧文氏菌 chalcidae（Erwinaze）），使得裸药物具有高度免疫原性。目前的护理标准是 Elspar的PEG化版本称为OnCaspar。虽然PEG化减少，但不消除 使用这些药物的免疫学挑战，另一个引起毒性的因素仍然存在-这是他们的L- 转氨酶协同活性。因此，OnCaspar仅限于ALL，即使其用于治疗成人ALL患者， 非常有限。值得注意的是，L-天冬酰胺酶相关的副作用阻止了这种独特的抗癌药物在癌症患者中的使用。 其它血液恶性肿瘤（例如急性髓性白血病）和实体瘤（例如胰腺癌）， 尽管有强有力的证据表明L-天冬酰胺酶在治疗这些癌症中是有效的。因此有 对免疫原性降低且缺乏L-天冬酰胺酶的L-天冬酰胺酶的明确未满足的需求 协同作用最近，我们鉴定了豚鼠L-天冬酰胺酶（GpA），其具有所需的低 KM性质的临床疗效和表现出体内肿瘤细胞杀伤。值得注意的是，我们还发现GpA 缺乏引起毒性的L-丙氨酸氨基转移酶共活性。与人L-半乳糖苷酶具有约70%的序列同一性， 天冬酰胺酶，GpA的免疫原性应低于细菌酶，仅占~25% 与人类酶的序列一致性。我们最近发现了主要的生物GpA 369，它是一种稳定的 和包含催化结构域的GpA的活性C-末端截短。在这里，我们将执行所需的 需要进行研究才能将这种新型酶药物带给患者。在目标1中，我们将其序列同一性增加至 使用结构指导的方法对人同源物进行修饰，并确定最佳聚乙二醇化策略。目标2将 确定来自目标1的前3个优化导联的药代动力学特性，并确认其抗- 在人类ALL小鼠模型中的癌症疗效。在目标3中，最佳变体（PK和抗- 癌症疗效）将进行毒性研究，首先在小鼠中进行，然后在大鼠中进行更广泛的研究， 狗最后，目标4将在一种新的小鼠模型中评价我们的酶药物的免疫原性， 重建人体免疫系统，并将我们的药物与OnCaspar进行比较。这些研究将为我们带来 到了提交IND申请在患者中测试这种新型L-天冬酰胺酶的时候。影响是 预计将扩展到ALL以外，因为我们的L-天冬酰胺酶变体的安全性改善将使 它在严重缺乏有效选择的多种癌症中的应用，并且强有力的数据表明， L-天冬酰胺酶药物将是有效的，但由于目前L-天冬酰胺酶的不可接受的毒性特征而未使用。 天冬酰胺酶的选择。

Project Summary: The goal of this proposal is to perform IND-enabling studies of a significantly safer variant of the anti-cancer biologic drug L-asparaginase. L-asparaginases are enzyme drugs that act to deplete the amino acid asparagine from the blood. Due to toxicity, which is especially pronounced in adults, L- asparaginase treatment is limited to acute lymphoblastic leukemia (ALL), a cancer of white blood cells. One source of toxicity of L-asparaginases is due to their bacterial origin (either from E. coli (Elspar) or Erwinia chrysanthemi (Erwinaze)), making the naked drugs highly immunogenic. The current standard of care is a PEGylated version of Elspar called Oncaspar. While PEGylation reduces, but does not eliminate the immunological challenge of using these drugs, the other toxicity-causing factor remains - this being their L- glutaminase coactivity. Therefore, Oncaspar is limited to ALL, where even its use to treat adult ALL patients is highly limited. Of note, L-asparaginase-associated side effects prevent the use of this unique cancer drug in other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. pancreatic cancer), despite strong evidence that L-asparaginases would be effective in treating those cancers. Hence, there is a clear unmet need for an L-asparaginase with reduced immunogenicity and that lacks L-glutaminase coactivity. Recently, we characterized a guinea pig L-asparaginase (GpA) that possesses the required low KM property for clinical efficacy and that exhibits in vivo tumor cell-killing. Notably, we also discovered that GpA is devoid of the toxicity-causing L-glutaminase co-activity. With ~70% sequence identity to human L- asparaginase, GpA should be less immunogenic compared to the bacterial enzymes that share only ~25% sequence identity with the human enzyme. We recently identified the lead biologic GpA369 which is a stable and active C-terminal truncation of GpA comprising the catalytic domain. Here we will perform the required studies required to bring this novel enzyme drug to patients. In Aim 1 we will increase its sequence identity to the human homolog using a structure-guided approach and identify the optimal PEGylation strategy. Aim 2 will determine the pharmacokinetic properties of the top 3 optimized leads from Aim 1, as well as confirm their anti- cancer efficacy in a human ALL mouse model. In Aim 3, the top variant (optimal combination of PK and anti- cancer efficacy) will proceed to toxicity studies, first in mice, followed by more extensive studies in rats and dogs. Finally, Aim 4 will evaluate the immunogenicity of our enzyme drug in a novel mouse model that has a reconstituted human immune system, and compare our drug to Oncaspar. Together, these studies will bring us to the cusp of submitting an IND application for testing this novel L-asparaginase in patients. Impact is predicted to extend beyond ALL, since the improved safety profile of our L-asparaginase variant would enable its use in multiple cancers for which effective options are sorely lacking and which strong data suggests that an L-asparaginase drug would be effective but is not used due to the unacceptable toxicity profile of current L- asparaginase options.