Biomaterial Scaffolds for Ex Vivo and In Situ CAR-T Cell Production

项目摘要 尽管嵌合抗原受体（CAR）-T细胞疗法在临床上取得了前所未有的成功， 广泛的应用受到冗长且劳动密集型的离体制造过程的限制 在：（i）高达50万美元的治疗成本非常高;（ii）输注CAR-T的延迟数周或数月 （iii）异质性组成和终末细胞 由于离体培养导致输注的CAR-T细胞分化，限制了CAR-T细胞植入， 坚持不懈克服这些限制的努力集中在封闭和自动化的制造设备上 以包含离体制造CAR-T细胞所需的劳动力，并且同种异体现成CAR-T细胞具有 已经提出了克服为每个患者制造CAR-T细胞的需要。尽管取得了重大 尽管在这一领域取得了巨大成就，但减少时间、成本和监管负担仍然是一个尚未得到满足的需求， CAR-T细胞疗法和显著减少或消除离体程序仍然是一个关键的未满足的需求。在 CAR-T细胞的体内产生将消除对离体程序的需要，防止终末细胞凋亡。 本发明的目的是提供一种用于体外扩增的CAR-T细胞的分化的方法，并确保自体T细胞的效力和寿命， 与广泛操作以防止排斥和移植的同种异体CAR-T细胞产品相比， 该提案中概述的研究开发了新的生物材料方法，以减少 体外产生CAR-T细胞的时间和努力，以增强CAR-T细胞的功效和体内持久性， 最后，通过完全在患者体内产生CAR-T细胞来完全消除离体操作。我们 提出生物材料支架展示抗CD 3/CD 28抗体并释放促增殖因子， 白细胞介素将介导T细胞的同时活化和病毒转导而无需离心 （spinoculation）或转导剂（retronectin，polybrene），并将促进离体遗传重编程 通过减少激活初始T细胞和用病毒载体转导它们的时间和费用， 我们接下来提出，直接植入接种有外周血单核细胞和CAR-1的支架， 编码病毒载体将促进CAR-T细胞释放到循环中，消除离体CAR-T分离 和增殖方案，以促进与较长的体内 坚持不懈最后，我们提出，通过包含包裹的T细胞吸引细胞因子， 植入的生物材料支架将通过募集宿主T细胞完全原位产生CAR-T细胞， 支架，用驻留的CAR编码病毒载体对募集的T细胞进行支架内重编程，和 释放重编程的CAR-T细胞。我们希望我们的研究结果将提供一个基础，为一般的细胞 治疗策略，并促进广泛的患者访问。除了在血液中的明显应用外， 癌症，这种合理的基于材料的细胞制造方法将被采用， 用于实体瘤和其他疾病的治疗性淋巴细胞。

PROJECT SUMMARY Despite unprecedented clinical success of chimeric antigen receptor (CAR)-T cell therapy against tumors, widespread application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) very high costs of therapy of up to half of a million dollars; (ii) delays of weeks or months to infuse CAR-T cells to patients with rapidly progressing disease; and (iii) heterogeneous composition and terminal differentiation of infused CAR-T cells as a result of ex vivo culture that limit CAR-T cell engraftment and persistence. Effort to overcome these limitations have focused on closed and automatic manufacturing devices to contain the labor needed to manufacture CAR-T cells ex vivo, and allogeneic off-the-shelf CAR-T cells have been proposed to overcome the need of CAR-T cell manufacturing for each single patient. Despite significant achievements in this space, reducing the time, costs and regulatory burden remains a deep unmet need in CAR-T cell therapy and significant reducing or eliminating ex vivo procedures remains a critical unmet need. In vivo generation of CAR-T cells would eliminate the need for ex vivo procedures, prevent the terminal differentiation of ex vivo expanded CAR-T cells and ensure the potency and longevity of autologous T cells as compared to allogeneic CAR-T cell products that are extensively manipulated to prevent rejection and graft- versus-host disease The research outlined in this proposal develops new biomaterials approaches to reduce the time and effort to produce CAR-T cells in vitro, to enhance CAR-T cell efficacy and persistence in vivo and, finally, to eliminate ex vivo manipulation entirely by generating CAR-T cells entirely within the patient. We propose that biomaterial scaffolds displaying anti-CD3/CD28 antibodies and releasing pro-proliferative interleukins will mediate simultaneous activation and viral transduction of T cells without centrifugation (spinoculation) or transduction agents (retronectin, polybrene) and will facilitate ex vivo genetic reprogramming of T cells by reducing the time and expense of activating naive T-cells and transducing them with viral vectors. We next propose that directly implanting scaffolds seeded with peripheral blood mononuclear cells and CAR- encoding viral vectors will promote release of CAR-T cells into circulation, eliminating ex vivo CAR-T isolation and proliferation protocols to promote a less differentiated cell phenotype associated with longer in vivo persistence. Finally, we propose that, through the inclusion of encapsulated T-cell attracting cytokines, implanted biomaterial scaffolds will generate CAR-T cells entirely in situ through recruitment of host T cells to the scaffold, in-scaffold reprogramming of recruited T cells with resident CAR-encoding viral vectors, and release of reprogrammed CAR-T cells. We expect that our results will provide a basis for a general cellular therapeutic strategy and promote widespread patient access. In addition to the obvious applications in blood cancers, this rational materials-based approach for cellular manufacturing will be adopted to program therapeutic lymphocytes in solid tumors and for other diseases.