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 -T）是一种令人兴奋的治疗方法，患者自身的免疫细胞在实验室中被改造以识别肿瘤。然后，这些细胞通过滴注的方式被送回患者体内，它们在患者体内生存和生长，并能发现并杀死癌细胞。car - t疗法对急性淋巴细胞白血病（ALL）患儿特别有效。大多数ALL患者对car - t有反应，但不到一半的患者在2年后仍然没有癌症。当car - t不再被检测到（即它们已经死亡），因此无法控制肿瘤时，癌细胞的返回（复发）经常出现。如果我们能让car - t持续更长时间（“持续”），我们就能提高不复发、保持无癌状态的患者数量。没有一个单一的因素使car - t在接受治疗的病人体内持续存在。如果CAR-T细胞“适合”自己，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“军队”来攻击癌症。然而，肿瘤细胞可以反击，并试图通过释放化学物质使可能攻击的CAR-T或其他免疫细胞丧失能力来生存。因此，car - t也需要能够在肿瘤细胞附近存活。被称为转录因子（tf）的小蛋白质自然存在于细胞中，被用作“控制面板”，可以同时快速打开许多开关。由于各种原因，它们被细胞用来快速改变细胞功能或行为。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数能使car - t更有效地杀死癌症或在患者体内存活更长时间的tf，但这些不太可能是最好的选择，因为它们是在高度人工的实验中发现的，不能很好地复制人类癌症。我们设计了一系列的实验来发现哪些tf可以用来使CAR-T细胞存活更长时间。1)我们将同时评估许多TFs 2) TFs可以增加或减少，科学家们大多描述了如果减少或删除TFs可以改善CAR-T功能。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。利用我们现有的技术，在CAR-T细胞中增加一种蛋白质更容易。因此，如果TF增加，我们将寻找使CAR-T工作得更好/持续时间更长的TF。3)我们将在复制肿瘤“不适宜”环境的动物模型中测试CAR-T和TF，因此我们可以确保CAR-T在这些条件下工作得更好，因此可能对患者也有更好的效果。在这个项目中，我们将首次系统地增加CAR-T细胞中800种可能的TFs中的一种的表达。将混合细胞（每个细胞仅增加1个TF的表达）注射到患有白血病的小鼠体内。能够使car - t持续存在的具有tf的特定细胞将比平常持续更长的时间，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，我们可以将这些鉴定出的tf引入到新的一批car - t细胞中，然后看看它们是否能在其他老鼠体内制造出更持久的car - t细胞，然后再在人类细胞中。我们的期望是，这项研究可以首先为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，所以我们期望使CAR-T细胞在ALL中更好地工作的tf可能与在其他类型的癌症中工作良好的tf不同。我们正在实验室开发许多动物模型，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的实验对于在ALL中创造更持久的CAR-T细胞是有用的，我们将在不同的癌症中重复这些实验，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.