Producing Humanized Therapeutics in Avian Egg White

描述(申请人提供)：该二期SBIR项目将测试在跨染色体禽蛋清中生产大量人类单抗的可行性。我们将使用在第一阶段创建和测试的全长卵粘蛋白基因组位点表达构建体来驱动单抗的重链和轻链cDNA的表达。利用这些构建物，我们培育出了能在蛋清中表达人类单抗的转基因母鸡。对该抗体进行了纯化，并对其抗原结合和效应功能进行了分析。这一分析结果支持了这样的结论，即产生的转基因抗体具有与哺乳动物细胞培养中产生的相同抗体相当的抗原结合和效应功能。这个第二阶段的项目还将利用改进的基因传递系统来产生完全跨染色体的禽类母鸡。我们最近产生了跨染色体创始人，展示了60MB人造染色体的生殖线传播。这些鸟的后代在每个细胞中都含有人工染色体的单一副本。这代表着禽类转基因方面的突破，到目前为止，这一突破一直受到逆转录病毒介导方法有效载荷能力有限的限制。我们将利用这种改进的禽类转基因技术将I期开发的两个基于卵粘蛋白的人源单抗表达载体输送到I期胚胎。来自GO、G1和G2禽类的转基因人单抗将被分析表达水平，纯化并鉴定生物活性和糖基化。我们预计，G2生产的鸡群将能够提供足够的人源单抗进行临床试验。

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.