Producing Humanized Therapeutics in Avian Egg White

项目描述（由申请人提供）：本二期SBIR项目将测试用转染色体禽蛋清生产大量人单克隆抗体的可行性。我们将使用在第一阶段创建和测试的全长卵泡样基因组位点表达构建体来驱动单克隆的重链和轻链cDNA的表达。利用这些构建体，我们培育出了在蛋清中表达人单克隆抗体的转基因母鸡。纯化该抗体并进行抗原结合和效应功能分析。这一分析结果支持这样的结论，即转基因产生的抗体具有抗原结合和效应功能，与哺乳动物细胞培养中产生的相同抗体相当。该二期项目还将利用一种改进的基因传递系统来产生完全的转染色体鸡。我们最近已经产生了一种60MB人工染色体的胚系传播的跨染色体创建者。这些鸟的后代在每个细胞中都含有人工染色体的单个副本。这是迄今为止受到逆转录病毒介导方法有效载荷能力有限限制的禽类转基因的一个突破。我们将利用这种改进的禽类转基因技术，将在I期胚胎中开发的两种基于卵母细胞的人单克隆抗体表达载体传递到I期胚胎。我们将分析从GO、G1和G2鸟中提取的转基因人单克隆抗体的表达水平、纯化并进行生物活性和糖基化鉴定。我们预计，G2生产群将能够提供足够的人单克隆抗体进行临床试验。

DESCRIPTION (provided by applicant): This phase II SBIR project will test the feasibility of producing large amounts of human monoclonal antibody into transchromosomic avian egg white. We will use a full length ovomucoid genomic locus expression construct created and tested in phase I, to drive expression of the monoclonal's heavy and light chain cDNA. Using these constructs we produced transgenic hens that expressed human monoclonal antibody into their egg whites. This antibody was purified and analyzed for antigen binding and effector function. The results of this analysis support the conclusion that the transgenic produced antibody exhibits antigen binding and effector function that is comparable to the same antibody produced in a mammalian cell culture. This phase II project will also utilize an improved gene delivery system to generate fully transchromosomic avian hens. We have recently generated transchromosomal founders that exhibit germline transmission of a 60MB artificial chromosome. The offspring of these birds contain single copies of the artificial chromosome in every cell. This represents a breakthrough in avian transgenesis which to this point has been restricted by the limited payload capacity of retroviral-mediated methods. We will utilize this improved avian transgenisis technique to deliver the two ovomucoid-based human monoclonal antibody expression vectors developed in phase I to stage I embryos. Transgenic human monoclonal antibody from GO, G1 and G2 birds will be analyzed for expression level, purified and characterized for bioactivity and glycosylation. We anticipate that the G2 production flock will be able to supply sufficient human monoclonal antibody to proceed to clinical trials.