Development and Pharmacology of Novel Lipidic rAHF

描述（由申请人提供）：蛋白质工程的进步导致了蛋白质作为治疗剂的发展。然而，一个常见的并发症是由于抗体反应而降低疗效。影响抗体反应的因素包括蛋白质聚集、蛋白质内的免疫原性序列和给药频率。该提案的主要目标是通过开发脂蛋白复合物来提高生物药物的治疗效果，这种复合物将降低免疫原性并减少蛋白质的清除，从而减少给药频率。因子VIII （FVIII）为研究这些方法提供了一个极好的机会，因为在15-30%的A型血友病患者中，外源性因子VIII会导致抑制性抗体反应的产生，使替代治疗复杂化。该项目的长期目标是开发FVIII的脂质复合物，积极调节免疫原性和清除。在之前的项目期间，合理的方法导致了fviii磷酸丝氨酸（PS）复合物的发展，在血友病A小鼠中注射后，对该蛋白的免疫反应降低，并改善了物理稳定性。利用PS与FVIII和钙离子的分子相互作用，开发了基于脂质的纳米/微颗粒，包括脂质体和用于FVIII递送的新型凝聚和纳米cochleate结构。本课题拟研究(1)FVIM-PS复合物介导免疫应答降低的机制(2)低密度受体相关蛋白和血管性血友病因子介导FVIII和FVIII- ps复合物清除的作用(3)抗体应答对FVIII和FVIII- ps复合物清除的影响。旨在了解FVIII-PS复合物药理学的研究将在血友病A小鼠模型和体外抗原呈递细胞（如树突状细胞和t细胞）中进行。我们建议研究免疫系统处理蛋白抗原的关键步骤，包括抗原呈递细胞对FVIII的摄取和加工，以及随后的t细胞的呈递和扩增。我们将研究PS在调节FVIII免疫原性中的作用（Specific Aim 1）。FVIII的药代动力学特性是复杂的，由于内在的蛋白质结合，我们将研究FVIII和FVIII- ps复合物在肝脏代谢和免疫系统介导下的处置。在具体目标2中，我们将研究PS结合和脂质分子组装对半衰期、曲线下面积和清除率等药代动力学参数的影响。最后，在具体目标3中，我们将研究抗体反应和FVIII处置的影响。从这些研究中获得的结果将有助于制定最佳剂量和有效的疾病管理和治疗。

DESCRIPTION (provided by applicant): Advances in protein engineering have led to the development of proteins as therapeutic agents. However, a common complication is the reduction of efficacy due to antibody response. Factors that influence antibody response include protein aggregation, immunogenic sequences within the protein and the frequency of administration. The broad objective of this proposal is to improve therapeutic efficacy of biopharmaceuticals by developing lipid-protein complexes that will reduce immunogenicity and decrease the clearance of the protein, thereby reducing frequency of administration. Factor VIII (FVIII) offers an excellent opportunity to investigate such approaches, as the administration of exogenous FVIII leads to development of inhibitory antibody responses in 15-30% of Hemophilia A patients, complicating replacement therapy. The long term goal of the project is to develop lipidic complexes of FVIII that positively modulate immunogenicity and clearance. During the previous project period, rational approaches led to the development of FVIIIPhosphoserine (PS) complexes that showed reduction in immune response against the protein following its injection in Hemophilia A mice and improved physical stability. The molecular interaction of PS with FVIII and calcium ions was exploited to develop lipid based nano/micro particulates including liposomes and novel condensed and nano-cochleate structures for FVIII delivery. In this proposal, we propose to investigate (1) the mechanism of reduction in immune response mediated by FVIM-PS complexes (2) the effect of low density receptor related protein and von Willebrand factor mediated clearance of FVIII and FVIII-PS complexes and (3) the effect of antibody response on clearance of FVIII and FVIII-PS complexes. The studies aimed at understanding the pharmacology of FVIII-PS complexes will be carried out in Hemophilia A mice model and in vitro with antigen presenting cells such as dendritic cells and T-cells. We propose to investigate key steps in the processing of protein antigen by the immune system in general, which include uptake and processing of FVIII by antigen presenting cells and subsequent presentation and expansion of Tcells. We will investigate the role of PS in modulating the immunogenicity of FVIII (Specific Aim 1). The pharmacokinetic properties of FVIII are complex due to intrinsic protein binding and we will investigate the disposition of FVIII and FVIII-PS complexes mediated by liver metabolism and immune system. In specific aim 2, we will investigate the effect of PS binding and lipid molecular assemblies on pharmacokinetic parameters such as half-life, area under the curve and clearance. Finally, in specific aim 3, we will investigate the effect of antibody response and disposition of FVIII. The results obtained from these studies will be useful to develop optimal dosing and efficient management of the disease and therapy.