Extending the in vivo half-life of a novel engineered mammalian asparaginase via its binding to human serum albumin

项目摘要：酶by Design(EBD)正在开发一种更安全的L-天冬酰胺酶(ASNase)，以最大限度地提高 这种独特药物的潜在临床应用。天冬氨酸氨基转移酶是一种能系统消耗L的酶类药物。 从血液中提取的天冬酰胺。在美国，第一线的ASNase是Oncaspar，一种聚乙二醇化的大肠杆菌 ASNase(ECA)。对Oncaspar不耐受的患者改用裸露的Erwinia ASNase(ERA，Erwinaze)。尽管 作为治疗儿童急性淋巴细胞白血病(ALL)的关键药物，目前FDA批准的副作用 ASNase在成年人中非常明显，因此很大程度上避免了使用它们。这些副作用也阻碍了使用 ASNase在其他血液系统恶性肿瘤(如急性髓系白血病)和实体肿瘤(如： 胰腺癌、卵巢癌或三阴性乳腺癌)，尽管有强有力的证据表明ASNase将是有效的 在治疗这些癌症方面。ECA/ERA的副作用源于：1)由于其细菌性而具有免疫原性 来源和II)它们的L-谷氨酰胺酶协同活性。将这种药物的使用范围扩大到成年所有患者和其他患者 适应症，需要一种具有(I)降低免疫原性，(Ii)缺乏L-谷氨酰胺酶联合... 活动性，结合(Iii)体内持久度长。为了减轻免疫原性，EBD正在开发 第一个哺乳动物的ASNase。与细菌ECA/Era形成鲜明对比，Eca/Era距离人类非常遥远 EBD正在开发豚鼠ASNase(GPA)，它在同源性上与人类ASNase更接近。 此外，通过遵循人源化过程，EBD增加了GPA与人类的序列同一性百分比 同源同源。总之，这种人源化的酶，被称为GpAhum，预计免疫原性要低得多 与细菌ASNase相比。这种酶的另一个优点是它本质上不含甘氨酸酶， 从而降低非靶向药物毒性。我们有大量的数据表明，在体内有很强的抗ALL效力 GpAhum毒性小。然而，GpAhum的半衰期(T1/2)并不适合临床使用。鉴于聚乙二醇化 提供了一种增加T1/2的解决方案，如Oncaspar所见，最近的临床数据报告了抗PEG抗体 在以前接受Oncaspar治疗的患者中，明确表示聚乙二醇化不是临床上可行的途径 任何新的ASNase。因此，为了实现增加的T1/2，我们转向人血清白蛋白(HSA)结合 通过将我们的ASNase融合到免疫中性的人血清白蛋白结合域(ABD)来实现这一技术。 该产品Abd-GpAhum具有出色的HSA，使父母GpAhum的血液循环时间增加了一倍以上 结合亲和力。在小鼠异种移植模型中的体内疗效证实了其优越的效力：1剂Abd-GpAhum 相当于3剂GpAhum，可在减少剂量频率和总剂量的情况下实现类似的疗效 注射的毒品数量。这预示着患者的累积毒性较小，药物相关治疗成本较低 以及医院相关工作人员的易用性。该提案将进一步评估Abd-GpAhum的抗ALL效果 并提供概念验证毒性数据，以证实Abd-GpAhum确实是可行的、安全的和有竞争力的 治疗候选药物，从而为通过支持IND的研究来推进它提供了理论基础。

PROJECT SUMMARY: Enzyme by Design (EbD) is developing a safer L-asparaginase (ASNase) to maximize the potential clinical applications of this unique drug. ASNases are enzyme drugs that systematically deplete L- asparagine from the blood. In the US, the 1st-line ASNase is Oncaspar, a PEGylated version of the E. coli ASNase (EcA). Patients intolerant of Oncaspar switch to the naked Erwinia ASNase (ErA, Erwinaze). Despite being key drugs in pediatric acute lymphoblastic leukemia (ALL), the side effects of current FDA-approved ASNases are so pronounced in adults that their use is largely avoided. These side effects also prevent the use of ASNases in other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. pancreatic, ovarian or triple-negative breast cancers), despite strong evidence that ASNases would be effective in treating those cancers. Side effects of EcA/ErA stem from i) their immunogenicity, due to their bacterial origin and ii) their L-glutaminase co-activity. To expand the use of this drug to adult ALL patients and to other indications, there is a need for an ASNase with (i) reduced immunogenicity, (ii) lack of L-glutaminase co- activity, combined with (iii) long in vivo persistence. To mitigate the immunogenicity, EbD is developing the first mammalian ASNase. In sharp contrast to the bacterial EcA/ErA, which are very distant from the human homolog, EbD is developing the guinea pig ASNase (GpA), which is much closer in identity to the human ASNase. Moreover, by following a humanization process, EbD increased GpA’s % sequence identity to the human homolog. Together, this humanized enzyme, referred to as GpAhum, is predicted to be much less immunogenic compared to the bacterial ASNases. An added advantage of this enzyme is that it is intrinsically GLNase-free, thereby reducing off-target drug toxicity. We have extensive data that shows strong in vivo anti-ALL potency of GpAhum with little toxicity. However, the half-life (t1/2) of GpAhum is not optimal for clinical use. Whereas PEGylation offers one solution for increasing the t1/2, as seen in Oncaspar, recent clinical data reporting anti-PEG antibodies in patients previously treated with Oncaspar made it clear that PEGylation is not a viable path to the clinic for any new ASNase. Therefore, to achieve an increased t1/2, we pivoted to human serum albumin (HSA)-binding technology via fusing our ASNase to an immunologically neutral human serum albumin binding domain (ABD). The product, ABD-GpAhum, more than doubled the parental GpAhum’s blood circulation time with excellent HSA binding affinity. In vivo efficacy in mouse xenograft models confirms its superior potency: 1 dose of ABD-GpAhum is equivalent to 3 doses of GpAhum, allowing similar therapeutic efficacy with reduced dosing frequency and total amount of drug injected. This predicts less accumulated toxicity in patients, lowered drug-related therapy cost and ease of use for hospital-related staffs. This proposal will further evaluate the anti-ALL efficacy of ABD-GpAhum and supply proof-of-concept toxicity data to confirm that ABD-GpAhum is indeed a viable, safe and competitive therapeutic candidate, thus providing the rationale for advancing it through IND-enabling studies.