| 英文摘要: | 1645031-Liu
In 2015, cancer caused at least 0.5 million deaths and 1.5 million new cases were diagnosed in the US. The adoptive transfer of large numbers of tumor-infiltrating T cells or genetically engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The existing methods to manufacture large quantities of such human T cells, however, have severe limitations of low efficiency, inconsistency and lack of sufficient quality control. This EAGER proposal aims to develop a novel human T cell biomanufacturing platform for large-scale, robust, and high-quality cellular production. The accomplishment of this study will provide not only the proof-of-concept but also the ready-to-use bioproduction platform for new means of T cell expansion for clinical immune cancer therapy. The novel technology employed in the rational production process engineering will also be able to provide guidelines and apply easily to the manufacturing of other therapeutic cells. Whereas the results and knowledge obtained in this study will be useful for both the biopharmaceutical industry and academic research, all cancer patients may benefit from the products of this research project.
The primary goal of this proposal is to develop an entirely new, metabolic cell process engineering (MCPE)-based, cellular biomanufacturing platform using stirred-tank bioreactor to produce reliable and reproducible large quantities of human T cells for immune cancer therapy, aiming to effectively produce more than 2,000 million T cells with high quality. The traditional T cell biomanufacturing presents several weaknesses: 1) low efficiency of mass transfer that often results in heterologous cellular metabolism, cell viability and product quality; 2) ineffective process parameter control that causes low robustness, reliability and scalability; and 3) lack of critical quality attributes in the early and middle stages of process development, limiting the application of quality by design. This project focuses on developing an innovative stirred-tank-based cellular biomanufacturing platform to produce reliable and reproducible large quantities of human T cells (or CAR T cells) for immune cancer therapy. Supported by Design of Experiment (DoE), proteomics and metabolomics will be applied to evaluate and determine the key bioproduction process parameters (such as stirred-tank parameters, media, supplements, etc.) to control T cell metabolism and cell growth. The oxygen transfer coefficient-based scale-up strategy will be developed to guide large-scale manufacturing of T cells, which will be validated using small- and medium- size tank bioreactors with scale-up factor of 10. In addition, at multiple key steps of the cellular bioproduction, the T cell quality control will be established via monitoring and evaluating cellular density, viability, T cell surface markers and functions. The existing T cell biomanufacturing in flask, LifeCell bag or Wave bag is limited by the weaknesses of lot-to-lot variation, heterologous product quality during scale-up, and low reproducibility. The proposed approach, i.e. MCPE-based fed-batch T cell production in stirred-tank bioreactor, that enables homogenous cell expansion, high cell density, high viability and good product quality in large-scale T cell manufacturing would be a major methodological advance for the field. Moreover, the systems biology approach will help advance the knowledge of host cell protein expression and intracellular metabolite profiling of human T cells under various culture conditions. In addition, the liquid activators in this proposed strategy will avoid heterologous suspension culture, improve cell growth efficiency, simplify manufacturing operation and reduce production cost. The critical scale-up factors learned from this application will guide future large-scale T cell biomanufacturing. Finally, the quality control at multiple stages of the process development will help identify potential product quality and process scale-up pain points during T cell bioproduction. To the PI's best knowledge, this is the first effort to rationally develop T cell bioproduction process via understanding the interaction between cellular metabolism and process parameters. |