Production Technology for Recombinant Intravenous Immunoglobulin

 描述（由申请人提供）：重组IVIg的生产技术组织：GigaGen Inc. PI：大卫S.约翰逊博士该SBIR I期项目的具体目标是开发天然库抗体蛋白表达系统，该系统将构成重组静脉内免疫球蛋白（rIVIg）治疗产品的基础。IVIg是从数千名献血者的血清中分离出来的蛋白质。FDA已批准IVIg治疗六种适应症，包括特发性（免疫性）血小板减少性紫癜（ITP）、川崎氏血管炎、B细胞慢性淋巴细胞白血病（CLL）和原发性免疫缺陷（橙子等人，2006年）。全球IVIg销售额为70亿美元，由于人口老龄化和不断扩大的标签外形式，每年增长8 - 10%（Taylor & Shapiro，2013）。不幸的是，由于供应链风险、杂质和污染以及批次间差异，目前的IVIg生产方法威胁到IVIg治疗的持续扩展。在这个I期项目中，我们将采取措施证明我们可以在稳定的酵母表达系统中表达GigaMune ™天然人类基因组DNA文库。类似于30年前的Genentech（Russo 2003），我们的主要技术创新是使用在人源化毕赤酵母生产系统中表达的天然IG库DNA文库（Li等人，2006年），以取代资源有限的生物药物与重组替代品。为了制备rIVIg，我们将首先使用GigaGen GigaMuneTM技术从> 1000名献血者中捕获并重新创建表达的IG库。GigaMuneTM使用先进的微流体技术和基因组学，从每个供体的数百万个单细胞中生成RT-PCR文库，其中包含天然IgG亚型以及重链和轻链之间的配对。然后，我们将在工程化毕赤酵母中稳定表达DNA库以产生大量多克隆工程化rIVIg蛋白产物。我们的rIVIg将具有天然的基因库、可工程化的内容、可编程的糖基化、低生产成本以及一致和可预测的生产。我们将通过执行以下任务来实现特定目标：（i）设计一个系统，用于亚克隆GigaLink（tm）DNA文库，其中天然IgG同种型完整;（ii）优化GigaLink（tm）DNA文库的递送和稳定性。 在毕赤酵母生产系统中展示;和（iii）使用下一代测序（NGS）和IG测定来评估几次细胞传代的均匀性和同种型含量。如果我们达到以下指标，我们将是成功的：（i）使用NGS显示当亚克隆并在酵母生产系统中稳定表达时，> 107多样性GigaLink（tm）文库的IG克隆频率得以维持（线性回归; a = 0.05，功效= 0.8）;和（ii）使用NGS和抗原结合测定来证明细胞传代和时间点之间<10%CV（单比例z检验; a = 0.05，功效= 0.8）。第一阶段将证明我们可以在酵母表达平台中可重复地产生高多样性的GigaLink（TM）蛋白质文库。在第二阶段，我们将采取措施建立GMP生产设施，并对我们的rIVIg制剂进行毒理学和药代动力学研究。首先，rIVIg将简单地替代常规IVIg，特别是对于抗体缺陷的患者（即，低丙种球蛋白血症或体液缺乏）。之后，我们对DNA文库内容进行工程改造的能力将开辟广泛的新应用，如IgA缺乏症和多克隆抗肿瘤治疗。

 DESCRIPTION (provided by applicant): Production Technology for Recombinant IVIg Organization: GigaGen Inc. PI: David S. Johnson, Ph.D. The Specific Aim of this SBIR Phase I project is to develop a natural repertoire antibody protein expression system that will form the basis of a recombinant intravenous immunoglobulin (rIVIg) therapeutic product. IVIg is a pool of proteins isolated from the sera of thousands of donors. The FDA has approved IVIg therapy for six indications, including idiopathic (immune) thrombocytopenic purpura (ITP), Kawasaki's vasculitis, B cell chronic lymphocytic leukemia (CLL), and primary immunodeficiencies (Orange et al., 2006). IVIg sales are $7 billion worldwide and growing at 8-10% per year, due to an aging population and ever-expanding off-label modalities (Taylor & Shapiro, 2013). Unfortunately, current methods for IVIg production threaten continued expansion of IVIg therapy because of supply chain risk, impurities and contamination, and batch-to-batch variation. In this Phase I project, we will take steps to demonstrate that we can express GigaMuneTM natural human repertoire DNA libraries a stable yeast expression system. Analogous to Genentech 30 years ago (Russo 2003), our primary technology innovation is to use natural Ig repertoire DNA libraries expressed in a humanized Pichia yeast production system (Li et al., 2006) to replace a resource-limited biological drug with a recombinant alternative. To make rIVIg, we will first use GigaGen GigaMuneTM technology to capture and re- create expressed Ig repertoires from >1000 blood donors. GigaMuneTM uses advanced microfluidics and genomics to generate RT-PCR libraries from millions of single cells per donor, with native IgG subtypes and pairing between heavy and light chain. We will then stably express the DNA repertoires en masse in engineered Pichia to produce massively polyclonal engineered rIVIg protein product. Our rIVIg will have natural repertoire genetics, engineerable content, programmable glycosylation, low production cost, and consistent and predictable production. We will accomplish the Specific Aim by performing the following tasks: (i) Engineer a system for subcloning GigaLink(tm) DNA libraries en masse with native IgG isotype intact; (ii) Optimize GigaLink(tm) DNA library delivery and stable display in a Pichia yeast production system; and (iii) Use next-generation sequencing (NGS) and Ig assays to assess several cell passages for uniformity and isotype content. We will be successful if we achieve the following metrics: (i) Use NGS to show that the Ig clone frequencies of the >107 diversity GigaLink(tm) libraries are maintained when subcloned and stably expressed in the yeast production system (linear regression; a=0.05, power=0.8); and (ii) Use NGS and antigen binding assays to demonstrate <10% CV between cell passages and time points (one-proportion z-test; a=0.05, power=0.8). Phase I will demonstrate that we can reproducibly produce high-diversity GigaLink(tm) protein libraries in a yeast expression platform. In Phase II, we will take steps to build a GMP production facility and perform toxicology and pharmacokinetic studies on our rIVIg preparations. At first, rIVIg will simply substitute for conventional IVIg, especially for patients who are deficient in antibodies (i.e., hypogammaglobulinemia or humoral deficiencies). Later, our ability to engineer the content of the DNA library will open up broad new applications, such as IgA deficiency and polyclonal anti-tumor therapeutics.