Immunocompetent in vivo CRISPR screening to identify key transcription factors which enhance persistence and efficacy of CAR-T cells in cancer

嵌合抗原受体T细胞(CAR-TS)是一种令人兴奋的治疗方法，患者自己的免疫细胞在实验室中被改造来识别肿瘤。然后，这些细胞通过滴注的方式回馈给患者，这些细胞在体内生长和生活的地方，可以发现并杀死癌细胞。CART对患有急性淋巴细胞白血病(ALL)的儿童尤其有效。大多数患者对CART有反应，但不到一半的患者在两年内仍未患上癌症。癌细胞的回归(复发)通常是当癌细胞不再被检测到时(即它们已经死亡)，因此无法控制肿瘤。如果我们可以让CART持续更长时间，我们就可以提高没有复发和保持无癌症的患者的数量。没有一个单一的因素可以让CART在接受治疗的患者身上持续存在。如果CAR-T细胞本身“合适”，它将更有可能持续存在：例如，它能够有效地杀死肿瘤并自我复制，形成一支攻击癌症的“军队”。然而，肿瘤细胞可以反击，并试图通过释放使CAR-T或其他可能攻击的免疫细胞丧失能力的化学物质来生存。因此，CAR-T还需要能够在肿瘤细胞附近生存。被称为转录因子(TF)的小蛋白质自然存在于细胞中，并被用作“控制面板”，同时快速打开许多开关。由于各种原因，它们被细胞用来迅速改变细胞的功能或行为。Tf在决定免疫细胞，如CAR-T细胞的行为方面至关重要。其他科学家已经描述了少量的转录因子，它们可以使CART更有效地杀死癌症，或者在患者身上存活更长时间，但这些不太可能是最佳选择，因为它们是在高度人工的实验中发现的，这些实验不能很好地复制人类癌症。我们设计了一系列实验，以揭示哪些TF可以用来使CAR-T细胞持续更长时间。1)我们将同时评估多个TF 2)TF可以增加或减少，科学家们大多描述了如果减少或删除，可以改善CAR-T功能的TF。这对CAR-T细胞疗法没有用处，因为降低用于治疗患者的细胞中的蛋白质水平既困难又昂贵。利用我们现在拥有的技术，在CAR-T细胞中增加蛋白质变得更容易。因此，我们将寻找在Tf增加的情况下使CAR-T工作得更好/更长时间的Tf。3)我们将在一个动物模型中测试CAR-T和Tf，该动物模型复制了‘不适宜居住的’肿瘤环境，因此我们可以确保CAR-T在这些条件下工作得更好，因此在患者身上也可能更好地工作。在这个项目中，我们将首次系统地增加&gt；800种可能的Tf中的一种在CAR-T细胞中的表达。一种混合的细胞(每个细胞的表达增加只有一种转铁蛋白)将被注射到患有白血病的小鼠体内。具有能够使CAR持续存在的TF的特定细胞将比通常持续更长的时间，然后这些细胞可以在实验室中进行分析和鉴定。为了验证我们的结果，我们可以将这些识别出的转录因子导入一批新的CAR-T中，然后观察它们是否在其他小鼠身上产生更持久的CAR-T，然后在人类细胞中产生。我们的期望是，这项研究可以在第一时间为ALL患者创造更好、更持久的CAR-T细胞，我们的目标是这项工作直接导致ALL的临床试验。然而，肿瘤是不同的，所以我们预计使CAR-T细胞在所有情况下都更好工作的转换因子可能与那些在其他类型的癌症中工作良好的转换因子不同。我们正在实验室中开发一些动物模型，以评估CAR-T细胞对抗不同类型癌症的能力。一旦我们证明这一系列实验对创造更持久的CAR-T细胞是有用的，我们将针对不同的癌症重复这些实验，目的是开发下一代更有效的CAR-T细胞。

Chimeric antigen receptor T cells (CAR-Ts) is an exciting treatment where a patient's own immune cells are engineered in the lab to recognise tumour. These cells are then given back to patients by drip where they live and grow in the body, and can find and kill cancer cells. CAR-Ts have been particularly effective in children with acute lymphoblastic leukaemia (ALL). Most ALL patients respond to CAR-Ts but less than half remain cancer free at 2 years. Return of cancer cells (relapse) is often seen when the CAR-Ts are no longer detectable (ie they have died out) so are not able to control tumour. If we could make CAR-Ts last longer ('persist'), we could improve the number of patients who do not relapse and remain cancer-free.There isn't one single factor which makes CAR-Ts persist in a treated patient. A CAR-T cell will be more likely to persist if it is 'fit' itself: for example, it is able to kill tumour efficiently and to replicate itself, forming an 'army' to attack the cancer. However, the tumour cells can fight back, and try to survive by releasing chemicals which incapacitate CAR-T or other immune cells that might attack. Thus CAR-Ts also need to be able to survive near tumour cells.Small proteins called transcription factors (TFs) exist naturally in cells and are used as 'control panels' to rapidly turn on lots of switches at the same time. They are used by the cell to rapidly change cell function or behaviour for a variety of reasons. TFs are critically important in deciding how immune cells, such as CAR-T cells, behave. Other scientists have described a small number of TFs that make CAR-Ts kill cancer more effectively or survive longer in patients, but these are unlikely to be the best options, as they were discovered in highly artificial experiments which don't replicate human cancer well. We have designed a series of experiments to uncover which TFs can be used to make CAR-T cells that persist longer. 1) We will assess many TFs at the same time 2) TFs can be increased or decreased and scientists have mostly described TFs that improve CAR-T function if they are reduced or deleted. This isn't useful for CAR-T cell therapy as it is difficult and expensive to reduce levels of proteins in cells used to treat patients. With the technology we have now, it is easier to increase a protein in a CAR-T cell. So we will look for TFs that make a CAR-T work better/ last longer if the TF is increased 3) We will test the CAR-Ts and TFs in an animal model that replicates the 'inhospitable' environment of tumour, so we can make sure the CAR-Ts work better in these conditions and therefore probably work better in patients too.In this project, we will for the first time, systematically increase the expression of one of >800 possible TFs in CAR-T cells. A mix of cells (each with increased expression of only 1 TF) will be injected into a mouse with leukaemia. The particular cells with TFs that are able to make CAR-Ts persist will last longer than usual - and these can then be analysed and identified in the laboratory. To check our results, we can then introduce those identified TFs into a new batch of CAR-Ts and then see if they make longer lasting CAR-Ts in other mice, and then in human cells. The expectation is that this research can create better and longer lasting CAR-T cells for patients with ALL in the first instance, and we aim that this work leads directly to a clinical trial in ALL. However, tumours are different so we expect that the TFs that will make a CAR-T cell work better in ALL may not be the same as those which work well in other types of cancer. We are developing a number of animal models in the lab to assess CAR-T cells against different cancer types. Once we have shown that these series of experiments are useful for creating longer lasting CAR-T cells in ALL, we will replicate these experiments for different cancers, with the aim of developing the next generation of more effective CAR-T cells.