Development of a Novel Method to Enhance Therapeutic Protein Production

描述(申请人提供)：任何蛋白质治疗练习的第一步都是蛋白质的表达。在某些情况下，这一步并不麻烦。然而，随着设计蛋白质数量的增加和翻译后修饰的要求，增强表达和增加蛋白质产量已经成为非常具有挑战性的任务。商品成本是一个重要因素。在某些情况下，治疗性蛋白质项目因为商品成本不合理而被放弃。公司花了12-18个月的时间来优化细胞系并提高重组蛋白的产量，这可能会让人感到惊讶。已经采取了几种方法来解决表达问题；然而，很可能没有一种单一的技术或程序能够解决难以表达的蛋白质问题。宿主的选择、培养条件和下游加工都会对产品蛋白质的质量和数量产生影响。Lifesensors之前已经描述了一种基于相扑的新型融合系统。将SUMO连接到非活性或低表达蛋白质的N-末端，可以显著提高蛋白质的功能，使蛋白质产量提高10-500倍。在原核生物中表达可获得完整的融合，该融合可通过相扑N端的各种亲和标签进行纯化。相扑融合在体外被一种高效的相扑蛋白酶切割，以产生具有所需N-末端的天然蛋白质。然而，类似的系统不适用于高等真核细胞，例如主要用于生产治疗性抗体和蛋白质的CHO。在这项建议中，我们描述了相关融合系统的开发，该融合系统将增强CHO细胞中难以表达的蛋白的表达和分泌。随着大量的工程抗体和治疗性蛋白作为临床候选药物的出现，蛋白质的表达已经成为一个真正的瓶颈。开发一种将蛋白质生产的质量和数量提高10倍的系统将是蛋白质疗法的一大进步。 与公共卫生相关：生产重组蛋白是一个昂贵的过程。越来越多的蛋白质被设计用于开发治疗产品。以合理的成本生产一些蛋白质变得越来越困难，以至于由于蛋白质生产的高成本而放弃了临床项目。我们已经提出了我们先前证明的融合蛋白的新用途。这样的策略可能会提高治疗发展中蛋白质表达的数量和质量。

DESCRIPTION (provided by applicant): The first step in any protein therapeutic exercise is expression of the protein. In some cases, this step is not troublesome. For increasing numbers of designer proteins and requirements for post-translation modifications, however, enhanced expression and increasing protein yield have become very challenging tasks. Cost of goods is an important factor. In some cases therapeutic protein projects are abandoned because of an unreasonable cost of goods. It may come as a surprise that companies spend 12-18 months to optimize the cell lines and improve the yield of recombinant proteins. Several approaches have been taken to overcome the expression problems; nevertheless, it is highly likely that no single technology or procedure will be able to overcome the difficult-to-express protein problem. Selection of host, culture conditions and downstream processing can all play a role in the quality and quantity of product protein. LifeSensors has previously described a novel SUMO based fusion system. Attachment of SUMO to N-termini of inactive or poorly expressed proteins has led to a remarkable improvement in the functionality of proteins and 10-500-fold enhancement in protein production. Expression in prokaryotes yields an intact fusion that is purified by virtue of a variety of affinity tags at the N-terminus of SUMO. SUMO-fusions are cleaved in vitro by a highly efficient SUMO protease to generate native protein with desired N-termini. However, a similar system is not available for higher eukaryotic cells ,e.g., CHO that are primarily used for production of therapeutic antibodies and proteins. In this proposal we describe the development of related fusion system that will enhance the expression and secretion of difficult-to-express protein in CHO cells. With the advent of large numbers of engineered antibodies and therapeutic proteins as clinical candidates, protein expression has become a real bottleneck. Development of a system that improves quality and quantity of protein production by 10x fold will be a great advance in protein therapeutics. PUBLIC HEALTH RELEVANCE: Production of recombinant protein is a costly process. Increasing numbers of proteins are engineered to develop therapeutic products. It is becoming difficult to produce some of the proteins at a reasonable cost to the point that clinical projects are abandoned due to high cost of protein production. We have proposed the novel use of our previously demonstrated fusion proteins. Such a strategy would likely increase the quantity and quality of protein expression in therapeutics development.