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

项目总结 尽管嵌合抗原受体(CAR)-T细胞疗法在抗肿瘤方面取得了前所未有的临床成功， 由于长时间和劳动密集型的体外制造程序，广泛的应用受到限制 在：(1)治疗费用非常高，高达50万美元；(2)延迟数周或数月注射CAR-T 快速发展的疾病患者的细胞；以及(Iii)异质成分和终末 限制CAR-T细胞植入和体外培养后输注的CAR-T细胞的分化 坚持不懈。克服这些限制的努力集中在封闭式和自动化制造设备上 为了遏制体外制造CAR-T细胞所需的劳动力，以及同种异体现成的CAR-T细胞 已被提出以克服为每个患者制造CAR-T细胞的需要。尽管意义重大 在这一领域取得成就，减少时间、成本和监管负担仍然是一个深层次的需求，尚未得到满足 CAR-T细胞治疗和显著减少或消除体外程序仍然是一个关键的未得到满足的需求。在……里面 体内产生CAR-T细胞将消除体外程序的需要，防止终末期 体外扩增的CAR-T细胞的分化及确保自体T细胞的效力和寿命 与被广泛操纵以防止排斥和移植的同种异体CAR-T细胞产品相比， 这项提案中概述的研究开发了新的生物材料方法来减少 在体外产生CAR-T细胞，提高CAR-T细胞在体内的有效性和持久性的时间和努力， 最后，通过在患者体内完全产生CAR-T细胞来完全消除体外操作。我们 提出展示抗CD3/CD28抗体并释放促增殖活性的生物材料支架 白介素会在不经离心的情况下介导T细胞的同时激活和病毒转导 (Spin Ococation)或转导剂(retronectin，Polybrene)，并将促进体外基因重新编程 通过减少激活原始T细胞并用病毒载体转导它们的时间和费用，可以减少T细胞的数量。 下一步，我们建议将外周血单个核细胞和CAR直接植入支架。 编码病毒载体将促进CAR-T细胞释放进入循环，从而消除体外CAR-T分离 和增殖方案，以促进分化较少的细胞表型与体内较长时间相关 坚持不懈。最后，我们建议，通过包含囊化的T细胞吸引细胞因子， 植入的生物材料支架将通过招募宿主T细胞来完全原位产生CAR-T细胞 用驻留的CAR编码病毒载体对招募的T细胞进行支架内重新编程，以及 释放重新编程的CAR-T细胞。我们希望我们的结果将为一般的蜂窝 治疗战略，促进患者的广泛接触。除了在血液中的明显应用之外 癌症，这种合理的基于材料的细胞制造方法将被采用来编程 实体肿瘤和其他疾病中的治疗性淋巴细胞。

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.