Production of Antibodies in Algae for Biodefense

描述(申请人提供)：单抗作为生物防御剂具有巨大的潜力，无论是在被动免疫、检测还是消毒方面。目前，这种潜力受到生产大量此类分子的成本和时间限制的限制。最常用的单抗生产系统依赖于在悬浮培养中培养的转化的中国仓鼠卵巢细胞(CHO)。CHO电池的生产是一个耗时的过程，要扩大到生产量，既麻烦又昂贵。为了应对大规模生产单抗所需的快速和廉价的系统，我们已经开始开发真核藻作为表达人抗体的生产平台。我们已经成功地开发了转化载体，可以在单细胞绿藻衣藻的叶绿体中表达多种重组蛋白，包括单链抗体、大单链和免疫球蛋白轻链和重链。根据这一建议，我们将在这些成功的基础上进行扩展，并演示从叶绿体和核质间组装两类全长人类抗体。这项研究的成功完成将为利用藻类作为一种快速产生大量重组人抗体的方法提供概念证明。具体来说，我们建议： 1)优化外源蛋白在藻叶绿体中的表达。 2)在藻类叶绿体中组装Fab和全长人源抗体。 3)表达并组装Fab抗体和全长抗体。 4)对藻类表达的抗体与哺乳动物表达的抗体进行生化和细胞分析。 5)规模化生产和纯化藻类表达抗体。

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.