Pharmacological and toxicological testing of a novel L-asparaginase

项目摘要：该提案的目标是对显著更安全的变种进行支持IND的研究 抗癌生物药物L-天门冬酰胺酶。L-天冬酰胺酶是一种酶类药物，可以消耗 血液中的氨基酸天冬酰胺。由于毒性，这一点在成年人中尤为明显，L- 天冬酰胺酶的治疗仅限于急性淋巴细胞性白血病(ALL)，一种白血球癌症。一 L-天冬酰胺酶的毒性来源是由于它们的细菌来源(来自大肠杆菌(Elspar)或欧文氏菌 菊花(Erwinaze)，使裸药具有高度的免疫原性。目前的护理标准是 埃尔斯帕的聚乙二醇化版本称为Oncaspar。虽然聚乙二醇化减少了，但并没有消除 使用这些药物的免疫挑战，另一个毒性因素仍然存在--这就是他们的L-- 谷氨酰胺酶协同活性。因此，Oncaspar仅限于ALL，即使它用于治疗成人ALL患者也是如此。 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 其他血液系统恶性肿瘤(如急性髓系白血病)和实体瘤(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方面是有效的。因此，有一个 对免疫原性降低且缺乏L-谷氨酰胺酶的L-天冬酰胺酶的明显未满足需求 同步性。最近，我们鉴定了一种豚鼠L-天冬酰胺酶(GPA)，它具有所需的低水平 KM具有临床疗效和体内肿瘤细胞杀伤力。值得注意的是，我们还发现GPA 没有引起毒性的L-谷氨酰胺酶共活性。与人类L有70%的序列同源性- 与仅占~25%的细菌酶相比，天冬酰胺酶，GPA的免疫原性应该较低 与人类酶的序列同源性。我们最近鉴定出铅生物GpA369是一种稳定的 以及包含催化结构域的GPA的活性C末端截断。在这里，我们将执行所需的 将这种新型酶药物带给患者所需的研究。在目标1中，我们将其序列同一性增加到 使用结构指导的方法进行人类同源物分析，并确定最佳的聚乙二醇化策略。目标2将 测定来自目标1的前3个优化导联的药代动力学特性，并确认它们的抗肿瘤活性 癌症在人类全鼠模型中的疗效。在目标3中，最上面的变体(PK和反-K的最佳组合 癌症疗效)将进行毒性研究，首先在小鼠身上，随后在大鼠和 狗。最后，目标4将在一种新的小鼠模型中评估我们的酶药物的免疫原性，该模型具有 重组的人体免疫系统，并将我们的药物与Oncaspar进行比较。总之，这些研究将为我们带来 即将提交IND申请，在患者中测试这种新型L-天冬酰胺酶。影响是 预计将超过所有这些，因为我们L-天冬酰胺酶变体的安全状况得到改善，将使 它在多种癌症中的应用严重缺乏有效的选择，而强有力的数据表明， L-天冬酰胺酶药物将是有效的，但由于目前L不可接受的毒性概况而不被使用- 天冬酰胺酶选项。

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.