Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Jacob Bridge, Matthew J Johnson, Jihyun Kim, Sophia Wenthe, Joshua Krueger, Bryce Wick, Mitchell Kluesner, Andrew T Crane, Jason Bell, Joseph G Skeate, Branden S Moriarity, Beau R Webber
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Abstract

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-γδ T cells in support of therapeutic application. Engineered CAR-γδ T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.
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用于免疫疗法的多克隆 γδ CAR-T 细胞的高效多重非病毒工程和扩增
γδ(γδ)T 细胞具有独特的能力,能识别转化细胞和病原体感染细胞上有限的非肽、非 MHC 相关抗原。除了缺乏异源活性外,γδ T 细胞还表现出不同于其他淋巴细胞亚群的特性,这促使人们对将其开发为现成的细胞免疫疗法产生了浓厚的兴趣。然而,γδ T 细胞在血液循环中的丰度低、异质性强、体外扩增方法有限以及基因修饰方法不完善等因素阻碍了γδ T 细胞的基础研究和临床应用。在这里,我们采用了一种无喂养器、可扩展的方法,在体外制造多克隆、非病毒修饰、基因编辑的嵌合抗原受体(CAR)-γδ T 细胞,以支持治疗应用。改造的 CAR-γδ T 细胞在体外和体内都表现出很高的功能。多克隆CAR-γδ T细胞的纵向体内药代动力学分析发现了亚群对IL-15细胞因子铠装和多重碱基编辑的特异性反应。我们的研究结果为多克隆 CAR-γδ T 细胞的基因修饰提供了一个强大的平台,并为进一步确定协同作用以及离散、工程化 CAR-γδ T 细胞亚群对体内疗效的贡献提供了独特的机会。
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