PDF(Pubmed)
Abstract：
Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform (\"Artificial Targets\") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.
摘要：
使用嵌合抗原受体（CAR）T细胞的癌症免疫疗法具有巨大的潜力，并证明了针对多种恶性肿瘤的临床疗效。研究和开发正在兴起，以加深对CART细胞功效的认识，并扩展这种新疗法的治疗潜力。为此,CART细胞的功能表征在基础研究和治疗开发的连续阶段中起着核心作用，随着标准化需求的增加。CART细胞的功能表征通常通过评估关键效应子功能来实现。在与表达靶抗原的细胞系共培养之后。然而,目标细胞系的使用带来了一些限制，包括细胞适应性的改变，由于细胞的处理和培养而导致的代谢状态或遗传漂移，这将增加变异性，并可能导致不一致的结果。此外，目标细胞系的使用可能是工作和时间密集的，并由于T细胞的同种异体反应而引入了重要的背景。为了克服这些限制,我们开发了一种基于合成珠子的平台（“人工靶标”）来表征体外CART细胞功能。这些合成微粒可以特异性诱导CAR-T细胞活化，如通过CD69和CD137(4-1BB)上调测量。此外,与人工靶标的接合导致诱导CART细胞的多种效应子功能，模拟由靶细胞系触发的反应，包括细胞毒性活性，通过暴露CD107a（LAMP-1）评估，细胞因子的表达和分泌，以及细胞增殖。重要的是,与靶细胞相反，人工靶刺激显示非特异性CAR-T细胞增殖有限。最后,人工靶标显示了接合多种共刺激分子的灵活性，这些共刺激分子可以协同增强CART细胞功能，并代表了调节CART细胞反应的强大工具。总的来说，我们的结果表明，人工靶标可以特异性地激活CART细胞，以实现必要的效应功能，这可以显着推进CART细胞功能评估的标准化，从早期开发到临床应用。
