Production of Recombinant Eukaryotic Ferroxidases as Protein Therapeutics

描述（由申请人提供）：在真核生物内管理铁需要一种或多种对作为底物的亚铁Fe II具有特异性的多铜氧化酶的活性。这些酶被称为铁氧化酶。它们的缺乏导致的表型范围从铁依赖性酶的活性下降，如在酵母中，到人类和其他哺乳动物的神经变性，这是由于铁沉积在大脑中，hCp缺乏引起的。这些铁氧化酶中的两种已经被很好地表征：来自酵母酿酒酵母的Fet 3蛋白，yFet 3 p，和人铜蓝蛋白，hCp。在人类中，Cp基因中的遗传性病变导致aceruloplasminemia（AC），即血液和中枢神经系统（CNS）中hCp活性的缺乏。前一种缺陷导致铁转铁蛋白的下降，从而导致向外周组织递送Fe的下降。尽管后一种下降，但Fe在CNS中积累，并可能支持导致神经元细胞死亡的化学作用，这是一种可能也涉及其他神经退行性疾病的机制。该R21申请的目的有两个：1）开发高通量、基于哺乳动物细胞的重组hCp生产; 2）开发将yFet 3 p和rhCp转化为治疗全身性铁的有效蛋白质治疗剂的策略。建立hCp的高容量表达系统的理由有两个：1）提供野生型hCp用于其作为蛋白质治疗剂的开发，2）提供AC突变蛋白用于随后阐明其分子缺陷。我们认为，这一见解将是重要的，原因有两个：1）它将导致对hCp机制的更好理解，2）在某些情况下，它将提出抑制体内给定缺陷的小分子策略。我们的高通量蛋白质生产将在连续瞬时转染条件下使用悬浮的HEK 298 EBNA 1细胞，该系统在生物技术和制药行业中应用最广泛。此外，我们将在无动物源（AOF）条件下生产这些蛋白质，这是药物开发中越来越多的要求。将铁氧化酶开发成有效的蛋白质治疗剂的基本原理是显而易见的;它们具有抑制铁的管理不善的潜力，铁的管理不善导致系统性功能性（非营养性）铁缺乏，特别是神经元细胞死亡。我们的蛋白质治疗开发策略有两个步骤：1）通过PEG化（聚乙二醇缀合）稳定循环中的yFet 3 p（以及随后的rhCp），而不损害铁氧化酶活性; 2）通过形成血脑屏障（BBB）的毛细血管内皮细胞中发现的转铁蛋白受体（TfR）将该蛋白质靶向CNS。聚乙二醇化目前正在使用中（例如干扰素药物，Pegasus和PegIntron），TfR，“特洛伊木马”策略已成功用于具有小分子和蛋白质治疗剂的模型系统中。我们将使用AC的aceruloplasminenic Cp-/-小鼠模型来测试我们的蛋白质治疗剂在恢复血液铁平衡方面的功效，使用高度道德的方案，不涉及放射性、麻醉或术后应激。铁是一种必需但有毒的营养素;它在大脑中的正常代谢的破坏与许多神经退行性疾病有关。这种联系的一个明显的例子是在遗传性疾病，aceruloplasminemia中发现的，其中患者缺乏酶，铜蓝蛋白，正常铁代谢所必需的酶的活性。这项研究的长期目标是确定患者的铜蓝蛋白中有什么缺陷，并开发一种基于蛋白质的药物，可以补充患者缺乏的铜蓝蛋白活性。

DESCRIPTION (provided by applicant): Managing iron within a eukaryote requires the activity of one or more multicopper oxidases specific towards ferrous iron, FeII, as substrate. These enzymes are called ferroxidases. Their absence leads to phenotypes that range from a decline in the activity of iron-dependent enzymes, as in yeast, to a neurodegeneration in humans and other mammals that is due to the Fe-deposition in the brain that hCp deficiency causes. Two of these ferroxidases have been well characterized: the Fet3 protein from the yeast Saccharomyces cerevisiae, yFet3p, and human ceruloplasmin, hCp. In humans, genetic lesions in the Cp gene lead to aceruloplas- minemia (AC), a lack of hCp activity in the blood and in the central nervous system (CNS). The former deficiency causes a decline in iron transferrin leading to a decline in the delivery of Fe to peripheral tissues. De- spite this latter decline, Fe accumulates in the CNS and likely supports the chemistry that leads to neuronal cell death, a mechanism perhaps involved in other neurodegenerative disorders as well. The objective of this R21 application is two-fold: 1) to develop high throughput, mammalian cell-based recombinant hCp production; and 2) to develop strategies for converting yFet3p and rhCp into pharmacologically efficacious protein therapeutics in the managing of systemic iron. The rational for establishing a high-volume expression system for hCp is two-fold: 1) to provide wild type hCp for its development as a protein therapeutic and 2) to provide AC mutant proteins for the subsequent elucidation of their molecular defects. We believe that this insight will be significant for two reasons: 1) it will lead to a greater understanding of the hCp mechanism in general and 2) in some cases, it will suggest small molecule strategies for suppressing a given defect in vivo. Our high-throughput protein production will use HEK298 EBNA1 cells in suspension under conditions of continuous transient transfection, the system most widely used in the biotech and pharmaceutical industries. Furthermore, we will produce these proteins under animal origin free (AOF) conditions as increasingly required in pharmaceutical development. The rationale for developing ferroxidases into effective protein therapeutics is obvious; they have the potential to suppress the mismanagement of iron that leads to a functional (not nutritional) iron deficiency systemically and to a neuronal cell death specifically. Our protein therapeutic development strategy has two steps: 1) stabilize yFet3p (and subsequently, rhCp) in the circulation by PEGylation (polyethylene glycol conjugation) without compromise of ferroxidase activity; 2) target this protein to the CNS via the transferrin receptor (TfR) found in the capillary endothelia that form the blood brain barrier (BBB). PEGylation is now in use (e.g. the interferon drugs, Pegasus and PegIntron) and the TfR, "Trojan Horse" strategy has been used successfully in model systems with both small molecule and protein therapeutics. We will use the aceruloplasminenic, Cp-/- mouse model of AC to test the efficacy of our protein therapeutics in restoring blood iron balance using a highly ethical protocol involving no radioactivity, anesthesia or post-procedure stress. Iron is an essential yet toxic nutrient; a break-down in its normal metabolism in the brain is linked to many neurodegenerative diseases. A clear example of this connection is found in the genetic disorder, aceruloplasminemia, in which the patient has a deficiency in the activity of the enzyme, ceruloplasmin, an enzyme essential for normal iron metabolism. The long-term goals of this research are to determine what is defective in the patients' ceruloplasmin protein and to develop a protein-based drug that can supplement the ceruloplasmin activity that the patients lack.