Production of Antibodies in Algae for Biodefense

描述（由申请人提供）：单克隆抗体（mAb）作为生物防御剂具有很大的潜力，无论是在被动免疫、检测还是消毒中。目前，这种潜力受到大量生产这些分子的成本和时间限制的限制。最常用的单克隆抗体生产系统依赖于在悬浮培养物中生长的转化的中国卵巢细胞（CHO）。在CHO细胞中的生产是一个耗时的过程，其繁琐且昂贵，难以扩大到生产量。为了响应对用于大规模生产mAb的快速且廉价的系统的需求，我们已经开始开发真核藻类作为用于表达人抗体的生产平台。我们已经成功地开发了转化载体，其允许在单细胞绿色衣藻（Chlamydiumreinhardtii）的叶绿体中表达多种重组蛋白，包括scFv、大单链以及IgA的轻链和重链。在这个建议下，我们将扩大这些成功，并证明装配两类全长人抗体从叶绿体和核质隔室。这项研究的成功完成将为使用藻类作为快速生产大量重组人抗体的方法提供概念证明。具体而言，我们建议： 1)优化异源蛋白在叶绿体中的表达。 2)在藻类叶绿体中组装Fab和全长人抗体。 3)表达并组装来自核质区室的Fab和全长抗体。 4)对藻类表达的抗体进行生物化学和基于细胞的分析，并与哺乳动物表达的抗体进行比较。 5)用于藻类表达的抗体的生产和纯化的放大。

DESCRIPTION (provided by applicant): Monoclonal antibodies (mAbs) have great potential as biodefense agents, whether in passive immunization, detection or disinfection. At present this potential is limited by the cost and time constraints in producing large quantities of these molecules. The most commonly used systems for monoclonal antibody production rely on transformed Chinese Hamster Ovary cells (CHO) grown in suspension culture. Production in CHO cells is a time-consuming process that is cumbersome and expensive to scale up to production volumes. In response to the need for a rapid and inexpensive system for the large scale production of mAbs, we have begun to develop eukaryotic algae as a production platform for the expression of human antibodies. We have successfully developed transformation vectors that allow for the expression of a variety of recombinant proteins, including scFvs, large single chain, and light and heavy chains of IgAs in the chloroplast of the unicellular green alga, Chlamydomonas reinhardtii. Under this proposal, we will expand upon these successes and demonstrate assembly of two classes of full-length human antibodies from both the chloroplast and nucleo-cytoplasmic compartments. Successful completion of this study should provide the proof of concept for using algae as a rapid means to produce large quantities of recombinant human antibodies. Specifically we propose to: 1) Optimize heterologous protein expression in alga chloroplast. 2) Assemble a Fab and full-length human antibody in algal chloroplast. 3) Express and assemble a Fab and full-length antibody from the nucleo-cytoplasmic compartment. 4) Carry out biochemical and cell based analysis of algal expressed antibodies in comparison with their mammalian expressed counterparts. 5) Scale up for production and purification of algal expressed antibodies.