Manufacturing of Growth Factors to Enable Cardiopoietic Stem Cell Therapy for Hea

描述（由申请人提供）：在心力衰竭患者的动物模型和I/II期临床试验中，造血细胞治疗显著改善了左心室功能，减弱了病理性重构，改善了功能结局。目前正在进行III期临床试验。在这种治疗方法中，骨髓来源的间充质干细胞通过暴露于促心性生长因子的混合物（包括激活素a、转化生长因子b1和骨形态发生蛋白4）进入心脏干细胞，这是临床制造中成本最大的单一成分。这些细胞因子都属于TGF-b超家族，对心脏干细胞的扩增和分化至关重要。然而，由于复杂的蛋白水解后修饰和在中性pH下的溶解度差，目前所有制造工艺的产率都很低，因此大批量生产是不经济的，而且不易获得。目前，商业产品是在非人类细胞系统（如R&D systems和PeproTech的大肠杆菌、SF9、CHO）或HEK293细胞（如HumanZyme和StemR&D的）中短暂生产的，仅作为研究用途的试剂。今天，世界上没有任何地方有能力生产这些必需的TGF-b超家族细胞因子，其规模和质量将满足未来大规模临床离体处理心脏和其他人类干细胞产品的要求。目前，这些高需求的细胞因子价格昂贵，每毫克6540- 9800美元，严重阻碍了干细胞治疗的临床规模处理。我们一直致力于开发一种具有成本效益和可扩展的表达系统，从稳定的HEK293SH细胞中产生真实的、重组的TGF-b超家族细胞因子，目前的人类细胞或非人类细胞表达系统要么不能生产，要么不能经济地生产。专利技术包括HEK293SH，一种选择的高产HEK293细胞系，适合在无血清和化学定义的培养基中悬浮生长，优化的人类启动子和信号肽，翻译后处理的优化，以及这些低溶解性蛋白的专有纯化和稳定方法。目前，Activin A、TGFb1和BMP4的产率比市面上任何其他产品高10-30倍，并且具有更高的稳定性、活性和纯度，从而大大降低了原材料和工艺人工成本。在本研究中，我们建议进一步优化生产，并开发大规模和高成本效益的生产和加工能力，以产生无血清和无异种TGFb超家族细胞因子，这将实现安全、稳健和高成本效益的心脏间充质干细胞治疗药物的体外加工。目标价格将从目前的6540- 9800美元/毫克降至100美元/毫克。

DESCRIPTION (provided by applicant): Cardiopoietic cell therapy significantly improved left ventricular function, blunted pathological remodeling, and functional outcomes in animal models and Phase I/II clinical trial of heart failure patients. There is ongoing Phase III trial. In this therapeutic approach, bone marrow-derived mesenchymal stem cells are engaged into cardiac stem cells by exposing to a cardiogenic cocktail of growth factors, including activin A, transforming growth factor-b1, and bone morphogenetic protein-4, which is the single largest cost component of clinical manufacturing. These cytokines all belong to the TGF-b superfamily, which are critical for cardiopoietic stem cell expansion and differentiation. However, due to the complex post-proteolytic modifications and poor solubility at neutral pH, yield from all the curren manufacturing process is very low, and as a result bulk volume is uneconomical and not readily available. Currently, commercial products are transiently produced in non-human cell systems (E coli, SF9, CHO, by, for example, R&D Systems and PeproTech) or HEK293 cells (by, for example, HumanZyme and StemR&D) as research-use only reagents. Today, there is no capacity anywhere in the world to produce these essential TGF-b superfamily cytokines at the scale and quality that will meet the requirements for future large-scale clinical ex vivo processing of cardiopoietic and other human stem cell products. Currently, these high-demand cytokines are costly, $6540-$9,800/mg, which severely hinders clinical scale processing of stem cell therapeutics. We have been focused on the development of a cost-effective and scalable expression system to produce authentic, recombinant TGF-b superfamily cytokines from stable HEK293SH cells that current human cell or non-human cell expression systems either cannot produce or cannot economically produce. The proprietary technology includes HEK293SH, a selected high-yield HEK293 cell line adapted to suspension for growth in serum-free and chemically defined medium, an optimized human promoter and signal peptide, optimization of post-translational processing, and proprietary purification and stabilization methods for these poor-solubility proteins. Currently, Activin A, TGFb1 and BMP4 are produced at >10-30 fold higher yield and exhibit greater stability, higher activity, and higher purity than any other products commercially available, resulting in profoundly lower raw material and process labor costs. In this study, we propose to further optimize the production and develop large-scale and cost-effective production and processing capabilities for generating serum-free and xeno-free TGFb superfamily cytokines, which will enable safe, robust, and cost-effective ex vivo processing of cardiopoietic mesenchymal stem cell therapeutics. The targeted price will be reduced from current $6540-$9,800/mg to $100/mg.