A protein-based method for the generation of allogeneic chimeric antigen receptor T-cells

嵌合抗原受体(CAR)T细胞疗法最近在美国和欧洲被批准用于治疗复发的B细胞恶性肿瘤，并有可能进一步应用于广泛的肿瘤类型。目前，大多数CAR T细胞产品都是为每个患者生产的自体定制产品。这有几个缺点：每个患者都必须接受白细胞分离；生产失败率约为10%；产品质量得不到保证；治疗有3-4周的滞后时间；无法应用生产规模经济。一个潜在的解决方案是来自健康捐赠者的同种异体CAR T细胞，可以提前制造、质量检查和冷冻保存，以便及时给任何潜在患者使用。然而，与供者无关的CAR T细胞通过供者T细胞受体(TCR)介导的移植物抗宿主病(TaGvHD)会导致严重或致命的移植物抗宿主病。为了提供同种异体CAR T细胞，必须防止TCR信号。到目前为止，同种异体CAR T细胞策略已经使用基因组编辑来删除TCR。事实上，我们是第一个描述从同种异体TALEN编辑的细胞中产生的抗CD19 CAR T细胞的临床应用。虽然已经看到了一些临床疗效，但需要一个复杂的多阶段制造工艺，非常严格地消耗TCR阳性T细胞。这一困难的制造消除了同种异体细胞在实用性/商品成本方面的大部分优势。自那以后，我们开发了一种无需基因组编辑的同种异体汽车T细胞制造的替代平台。通过将TCR特异性单链可变片段与高尔基保留信号(TCR-KDEL)与CAR一起共表达，TCR阴性的CAR T细胞可以通过单一的病毒转导而产生。此外，GMP兼容标记基因的额外共表达有助于在单一分选步骤中方便地高度严格地选择TCR阴性、CAR阳性的细胞。有了这项资助，我们建议在晚期B细胞恶性肿瘤患者中测试TCR-KDEL同种异体CAR T平台，使用我们中心目前正在进行几项研究的下一代CD19 CAR(CAT19)。虽然我们预计TCR-KDEL的使用不会局限于CD19 CAR，但使用CAT19进行测试可以直接与正在进行的自体CAR研究进行比较，并为潜在的未来研究提供概念证明。我们预计我们的方法可以降低成本并拓宽CAR T细胞治疗的途径。

Chimeric Antigen Receptor (CAR) T-cell therapy has recently been approved in the USA and Europe for the treatment of relapsed B-cell malignancies, with potential further application in a broad range of tumour types. Currently, most CAR T-cell products are generated as autologous bespoke products for each patient. This has several disadvantages: each patient must undergo leukapheresis; production failure rate is ~10%; product quality is not guaranteed; there is a 3-4 week lag time to treatment; and economies of manufacturing scale cannot be applied.A potential solution is allogeneic CAR T-cells from healthy donors, which could be manufactured in advance, quality-checked and cryopreserved for timely administration to any potential patient. However, unrelated donor CAR T-cells would cause severe or fatal graft-versus-host-disease (taGvHD) mediated by the T-cell receptor (TCR) of donor T-cells. To deliver allogeneic CAR T-cells, TCR signalling must be prevented.To date, allogeneic CAR T-cell strategies have used genome-editing to delete the TCR. Indeed, we were the first to describe clinical use of anti-CD19 CAR T-cells generated from allogeneic TALEN-edited cells. While some clinical efficacy has been seen, a complex multi-stage manufacturing process is required, with highly stringent depletion of TCR positive T-cells. This difficult manufacture obviates much of the advantage in terms of practicality / cost of goods for allogeneic cells.We have since developed an alternative platform for allogeneic CAR T-cell manufacturing without genome-editing. By co-expressing a TCR-specific single-chain variable fragment with a Golgi retention signal (TCR-KDEL) along with the CAR, TCR-negative CAR T-cells can be generated with a single viral transduction. Further, additional co-expression of a GMP compliant marker gene facilitates facile highly stringent selection of TCR-negative, CAR-positive cells in a single sorting step.With this grant, we propose to test the TCR-KDEL allogeneic CAR T platform in patients with advanced B-cell malignancies, using a next-generation CD19 CAR (CAT19) currently in several studies at our centre. While we do not expect TCR-KDEL usage to be limited to CD19 CAR, testing with CAT19 allows direct comparison with ongoing autologous CAR studies, and acts as a 'proof of concept' for potential future studies. We anticipate our approach could reduce cost and broaden access to CAR T-cell therapy.