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

制造生长因子以实现心脏造血干细胞治疗

• 批准号: 8766958
• 负责人: RIDONG CHEN
• 金额: $ 22.41万
• 依托单位: APT THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766958
• 关键词: Affect; Animal Model; Animals; BMP4; Bioreactors; Blood; Bone Marrow; Cardiac; Cell Culture Techniques; Cell Line; Cell Therapy; Cell physiology; Cells; Clinical; Clinical Trials; Complex; Development; Disease; Endotoxins; Ensure; Equipment; Escherichia coli; Exhibits; Future; Growth; Growth Factor; Guidelines; Health Expenditures; Heart failure; Human; Human Development; Laboratories; Left Ventricular Function; Mesenchymal; Mesenchymal Stem Cells; Methods; Modification; Myocardial; Natural regeneration; Patients; Peptide Signal Sequences; Persons; Phase; Phase III Clinical Trials; Price; Process; Production; Proteins; Reagent; Recombinants; Recruitment Activity; Research; Risk; Secure; Serum; Sialic Acids; Solubility; Stem cells; Suspension substance; Suspensions; System; Technology; Testing; Therapeutic; Transforming Growth Factors; Work; Xeno; activin A; bone morphogenetic protein 4; cost; cost effective; cytokine; functional outcomes; human stem cells; immunogenic; improved; in vivo; killings; manufacturing process; meetings; outcome forecast; promoter; public health relevance; research and development; stem cell therapy; success

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.

描述（由申请人提供）：在动物模型和心力衰竭患者的 I/II 期临床试验中，心肌细胞疗法显着改善左心室功能、减弱病理重塑和功能结果。 III 期试验正在进行中。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原性混合物而与心脏干细胞结合，这些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，这是单一最大的临床制造的成本组成部分。这些细胞因子均属于 TGF-b 超家族，对心脏生成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 下溶解度差，目前所有生产工艺的产率非常低，因此批量生产不经济且不易获得。目前，商业产品在非人类细胞系统（大肠杆菌、SF9、CHO，例如 R&D Systems 和 PeproTech）或 HEK293 细胞（例如，HumanZyme 和 StemR&D）中瞬时生产，作为仅供研究使用的试剂。如今，世界上任何地方都没有能力生产这些必需的 TGF-b 超家族细胞因子，其规模和质量无法满足未来大规模临床离体加工心脏生成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，为 6540-9,800 美元/毫克，严重阻碍了干细胞疗法的临床规模加工。我们一直专注于开发一种经济有效且可扩展的表达系统，以从稳定的 HEK293SH 细胞中产生真实的重组 TGF-b 超家族细胞因子，而当前的人类细胞或非人类细胞表达系统无法产生或无法经济地产生这些细胞因子。专有技术包括 HEK293SH（一种精选的高产 HEK293 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）、优化的人类启动子和信号肽、翻译后处理的优化以及专有的纯化和稳定方法。这些溶解度差的蛋白质。目前，Activin A、TGFb1 和 BMP4 的产量比任何其他市售产品高 10-30 倍，并且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。在本研究中，我们建议进一步优化生产并开发大规模且具有成本效益的生产和加工能力，以产生无血清和无异种的 TGFb 超家族细胞因子，这将使安全、稳健且具有成本效益的离体心肌间充质干细胞疗法的加工。目标价格将从目前的 6540-9,800 美元/毫克降至 100 美元/毫克。