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

描述（由申请人提供）：造血细胞疗法在心力衰竭患者的动物模型和I/II期临床试验中显著改善了左心室功能，减弱了病理性重塑和功能结局。目前正在进行III期试验。在这种治疗方法中，骨髓来源的间充质干细胞通过暴露于生长因子的心源性鸡尾酒（cardiogenic cocktail）而参与心脏干细胞，所述生长因子包括激活素A、转化生长因子-b1和骨形态发生蛋白-4，这是临床制造的单一最大成本成分。这些细胞因子都属于TGF-b超家族，其对心脏造血干细胞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下的溶解性差，所有当前制造工艺的产率都非常低，因此批量体积是不经济的并且不容易获得。目前，商业产品在非人细胞系统（大肠杆菌、SF 9、CHO，例如R&D Systems和PeproTech）或HEK 293细胞（例如HumanZyme和StemR&D）中瞬时生产，作为仅供研究使用的试剂。今天，世界上任何地方都没有能力以满足未来大规模临床离体处理心脏造血和其他人类干细胞产品的要求的规模和质量生产这些必需的TGF-β超家族细胞因子。目前，这些高需求的细胞因子是昂贵的，$6540-$9，800/mg，这严重阻碍了干细胞疗法的临床规模处理。我们一直专注于开发一种具有成本效益和可扩展的表达系统，以从稳定的HEK 293 SH细胞中生产真正的重组TGF-β超家族细胞因子，目前的人类细胞或非人类细胞表达系统不能生产或不能经济地生产。专有技术包括HEK 293 SH，一种选择的高产HEK 293细胞系，适合在无血清和化学成分确定的培养基中悬浮生长，优化的人启动子和信号肽，翻译后加工的优化，以及这些溶解性差的蛋白质的专有纯化和稳定方法。目前，激活素A、TGF β 1和BMP 4的产量比任何其他市售产品高10-30倍，并表现出更高的稳定性、更高的活性和更高的纯度，从而大大降低了原材料和工艺劳动力成本。在这项研究中，我们建议进一步优化生产，并开发大规模和具有成本效益的生产和加工能力，以产生无血清和无异种TGF β超家族细胞因子，这将使心脏造血间充质干细胞治疗剂的安全，稳健和具有成本效益的离体加工成为可能。目标价格将从目前的6540 - 9,800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.