The influence of co-stimulatory domains on the metabolic regulation of chimeric antigen receptor (CAR) T cell function

T细胞是免疫系统的重要组成部分，促进/设计T细胞反应目前处于癌症免疫治疗的前沿。嵌合抗原受体(CAR)T细胞疗法在治疗某些亚群的B细胞白血病或淋巴瘤方面显示出显著的临床疗效。CAR由针对肿瘤靶点的细胞外抗原结合区组成，与细胞内信号域相连，以信号传递T细胞激活和效应器功能。在识别抗原后，CAR分子发生聚集和固定，这导致CD3-Zeta(CD3z)链的磷酸化，类似于通过天然T细胞受体传递信号。这会触发T细胞增殖、细胞因子释放、代谢变化和细胞毒性；T细胞效应器反应。CAR T细胞疗法对白血病和淋巴癌非常有效，然而，到目前为止，对实体肿瘤的治疗结果令人失望。T细胞发挥全部效应功能(如增殖、细胞毒性)的能力关键取决于细胞能量的最佳利用和环境中营养物质的可用性。实体肿瘤中的肿瘤细胞可以通过控制肿瘤微环境(TME)中营养物质的可获得性来抵消这种免疫反应，从而导致治疗失败。T细胞共刺激可通过CD28和4-1BB传递。目前临床上的CAR T细胞携带共刺激结构域，称为第二代CAR，第三代CAR也已被开发出来，其中两个共刺激结构域作为CAR的一部分被放置在一条线上。令人失望的是，第三代汽车在临床上并没有表现出比第二代汽车更好的性能(1)。现在，我们的实验室已经生成了一个并行CAR(PCAR)结构，以优化同时提供CD28和4-1BB的共刺激信号，一个作为CAR的一部分，另一个作为近端但独立的嵌合共刺激分子(Muliaditan，Flaherty，Maher，Schurich，手稿正在审查中)。与目前临床应用的CAR T细胞相比，这些PCAR T细胞在体外和体内显示出更强的肿瘤杀伤能力。在体外，PCAR T细胞更能抵抗T细胞的衰竭和衰老，维持增殖和细胞因子的释放。我们假设PCAR T细胞功能的增强是由于T细胞代谢和线粒体功能的改善。我们的初步发现支持这一假说，表明在刺激后，PCAR T细胞与传统CAR细胞相比，具有显著更高的营养转运蛋白表达，并具有更高比例的功能性线粒体。PCAR T细胞吸收葡萄糖和铁等基本营养物质的能力增强，表明它们可以维持高速新陈代谢，有助于它们的功能和增殖。此外，它们线粒体功能的增强表明它们的寿命更长，对疲惫的抵抗力更强，这一过程我们以前已经证明在慢性病毒感染中至关重要(2)。我们相信，这些因素将有助于更有效的治疗性CAR T细胞产品，以抵御缺乏营养的肿瘤微环境。

T-cells are a vital component of the immune system and promoting/engineering T-cell responses is now at the forefront of cancer immuno-therapies. The development of chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical responses in treating certain subsets of B cell leukemia or lymphoma. CAR's are composed of an extracellular antigen-binding region against the tumour target linked to intracellular signalling domains to signal for T- cell activation and effector function. Upon recognising antigen, clustering and immobilisation of CAR molecules occurs which induces phosphorylation of the CD3-zeta (CD3z) chain akin to signalling via the natural T cell receptor. This triggers T-cell proliferation, cytokine release, metabolic changes and cytotoxicity; the T-cell effector response. CAR T- cell therapy has been highly effective in leukemic and lymphatic cancers, however, so far, has shown disappointing results for the treatment of solid tumours. The ability of a T-cell to display full effector functions (e.g. proliferation, cytotoxicity) crucially depends on optimal cellular energy utilisation and the availability of nutrients in the environment. Tumour cells in solid tumours can counteract this immune response by manipulating the availability of nutrients in the tumour microenvironment (TME), leading to treatment failure. T-cell co-stimulation can be delivered through both CD28 and 4-1BB. CAR T cells currently in the clinic carry either costimulatory domain, termed 2nd generation CAR, and 3rd generation CAR have also been developed in which both co-stimulatory domains were placed in line as part of the CAR. Disappointingly 3rd generation CAR did not show improved performance compared to 2nd generation CAR in the clinic (1). Now, our lab has generated a parallel-CAR (pCAR) construct to optimise costimulatory signalling delivering both CD28 and 4-1BB, one as part of the CAR and one as a proximal but independent chimeric costimulatory molecule (Muliaditan, Flaherty, Maher, Schurich, manuscript under review). These pCAR T-cells have shown enhanced tumour killing capacity in vitro and in vivo compared with CAR T-cells currently in the clinic. In vitro, pCAR T-cells have shown to be more resistant to T-cell exhaustion and senescence, sustain proliferation and cytokine release. We hypothesise that the increased functionality of pCAR T-cells is due to improved T-cell metabolism and mitochondrial function. Our preliminary findings have supported this hypothesis, showing that following stimulation, pCAR T-cells have significantly higher expression of nutrient transporters, and have a higher proportion of functional mitochondria when compared to conventional CAR counterparts. The increased ability of the pCAR T-cells to take up essential nutrients like glucose and iron suggests that they can sustain a high rate metabolism, contributing to their function and proliferation. Furthermore, the enhanced functionality of their mitochondria suggests increased longevity and resistance to exhaustion, a process we have previously shown vital in chronic viral infection (2). We believe these factors will contribute to a more effective therapeutic CAR T-cell product to withstand the nutrient lacking tumour microenvironment.