Engineering detours around the biologic barriers to allogeneic, iPSC-derived CAR T immunotherapy

工程绕开了同种异体、iPSC 衍生的 CAR T 免疫疗法的生物障碍

• 批准号: 10607952
• 负责人: Sang Pil Yoo
• 金额: $ 4.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607952
• 关键词: 3' Untranslated Regions; 3-Dimensional; Address; Affect; Allogenic; Antigens; Autologous; Biological; CAR T cell therapy; CD19 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR/Cas technology; Cell Differentiation process; Cell Maturation; Cell Therapy; Cells; Cellular biology; Cellular immunotherapy; Clinical; Data; Development; Engineering; Excision; Generations; Genes; Genetic; Genetic Transcription; Granzyme; Health Services Accessibility; Hematologic Neoplasms; Immunoglobulin Constant Region; Immunotherapy; In Vitro; Lymphoid; Malignant Neoplasms; Mature T-Lymphocyte; Methods; Modeling; Organoids; Pathway interactions; Patients; Pattern; Physiological; Pluripotent Stem Cells; Positioning Attribute; Rag1 Mouse; Receptor Cell; Receptor Signaling; Refractory; Research; Risk; Signal Transduction; Source; System; T cell differentiation; T cell therapy; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Technology; Thymus Gland; Transgenes; Tumor Antigens; cancer type; chimeric antigen receptor; chimeric antigen receptor T cells; clinical care; constitutive expression; cost; cytotoxicity; graft vs host disease; human pluripotent stem cell; improved; in vivo; individualized medicine; induced pluripotent stem cell; innovation; interest; manufacturing process; monolayer; multipotent cell; novel; prevent; receptor expression; receptor-mediated signaling; response; stem cell technology; stem cells; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has produced remarkable results in otherwise treatment- refractory hematological malignancies. Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individualized therapy and the inherent variability in T cell biology between patients, leading to unpredictable and inconsistent clinical responses. As a result, there is a growing interest in generating CAR T cells in vitro from an infinitely renewing, allogeneic source of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to genetic editing, providing multiple avenues to manipulate the function of the final T cell product. However, there are several biologic barriers to generating CAR T cells from hPSCs. First, expression of the CAR transgene early in T cell differentiation diverts development towards the innate lymphoid pathway instead of the conventional T lineage. Second, the removal of the endogenous T cell receptor (TCR) to prevent alloreactivity leads to a block in development, as maturing T cells can no longer undergo positive selection. This proposal seeks to overcome these challenges to generating non-alloreactive, stem cell-derived CAR T cells by deploying an innovative stage-specific expression strategy and a novel in vitro method to induce the differentiation of mature conventional T cells, the Artificial Thymic Organoid (ATO) system. The ATO is a first-in-class, in vitro method for efficiently generating mature, single positive (SP) CD8+ and CD4+ T cells from multiple stem cell sources, including hPSCs. Improving upon previously established systems, the ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have already demonstrated that innate fate diversion can be overcome by achieving delayed, stage-specific expression of the CAR transgene that is limited to mature T cells. In this proposal, Specific Aim 1 will build upon the delayed CAR expression model and evaluate how the disruption of the endogenous TCR affects CAR T cell development in the ATOs. Specific Aim 2 will evaluate two different methods of delivering exogenous positive selection signals to rescue development in the absence of the endogenous TCR. These methods include expression of a tonically signaling CAR or transiently expressing an exogenous TCR at the physiologically appropriate double positive (DP) T cell stage. The approaches developed in this proposal will enable the generation of hPSC-derived, non-alloreactive T cell therapy, ultimately reducing the cost and increasing the access to treatment for more patients in need.

项目总结/摘要 嵌合抗原受体（CAR）T细胞疗法在其他治疗中产生了显着的效果- 难治性恶性血液病目前，制造自体CAR T细胞的过程是 具有挑战性，因为需要重新生成每种个体化治疗，以及 患者之间的T细胞生物学，导致不可预测和不一致的临床反应。结果是 越来越多的人对从无限更新的同种异体人类来源体外产生CAR T细胞感兴趣， 多能干细胞（hPSC）。这种方法的一个优点是hPSC高度适合遗传修饰。 编辑，提供多种途径来操纵最终T细胞产物的功能。 然而，从hPSC产生CAR T细胞存在几个生物屏障。第一，表达 T细胞分化早期的CAR转基因使发育转向先天淋巴途径 而不是传统的T细胞谱系。第二，去除内源性T细胞受体（TCR），防止 同种异体反应性导致发育受阻，因为成熟的T细胞不再能够经历阳性选择。 该提案旨在克服这些挑战，以产生非同种异体反应性的干细胞衍生的CAR T。 通过部署创新的阶段特异性表达策略和一种新的体外方法来诱导细胞的增殖， 成熟常规T细胞的分化，人工胸腺类器官（ATO）系统。 ATO是一种一流的体外方法，可有效生成成熟的单阳性（SP）CD 8 + 和来自多种干细胞来源的CD 4 + T细胞，包括hPSC。改进以前建立的 系统，ATO可以稳健地支持正选择和成熟到SP阶段。我们的初步研究 我已经证明，先天的命运转移可以通过实现延迟的，阶段特定的， CAR转基因的表达仅限于成熟T细胞。在本提案中，具体目标1将建立在 延迟的CAR表达模型，并评估内源性TCR的破坏如何影响CAR T细胞 在ATO的发展。具体目标2将评价两种不同的外源性阳性药物输送方法 选择信号以在内源性TCR不存在的情况下拯救发育。这些方法包括 在生理上可接受的水平上瞬时表达紧张性信号传导CAR或瞬时表达外源性TCR。 适当的双阳性（DP）T细胞阶段。本提案中制定的方法将使 产生hPSC衍生的非同种异体反应性T细胞疗法，最终降低成本并增加免疫原性。 为更多有需要的患者提供治疗。