PO.IM01.06 · 免疫学
CD3阳性的异体CAR T细胞：一种具有持久性、BiTE捕获能力且无同种异体反应性的新型平台
CD3-positive allogeneic CAR T-cells: A novel platform with persistence, BiTE-capture, and no alloreactivity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
多种异体CAR T细胞疗法正处于临床试验中以评估安全性，其中许多依赖于基于CRISPR/Cas的基因组编辑。然而，Cas酶的随机修复机制提高了不期望的脱靶效应风险，为安全生成异体CAR T细胞带来了挑战。为减轻与治疗相关的致瘤风险，我们力图寻求一种不同于临床现有产品的替代性安全策略来制造异体CAR T细胞。我们开发了一种靶向T细胞受体β恒定区（TRBC）基因的CRISPR RNA（crRNA），它与AsCas12a Ultra酶联合使用，通过引发可预测的微同源介导的末端连接（MMEJ）DNA修复途径实现位点特异性编辑，从而减轻脱靶风险。采用靶向扩增子测序来评估脱靶和修复机制。使用免疫缺陷小鼠评估移植物抗宿主同种异体反应性以及CAR T的持久性。开展了历时逾3个月的体内肿瘤攻击研究。使用免疫组织化学和流式细胞术进行植入确认。有趣的是，在评估编辑后的T细胞时，我们分离出一个独特的T细胞群，其显示出TCR被破坏但仍保持CD3阳性且不引起体内同种异体反应性。这些CD3保留、可同种异体发挥功能的T细胞（CRAFT-cells）表现出与未编辑T细胞相似的生长速率，随后被用作平台以生产CD19靶向CAR T细胞和BAFF-R靶向CAR T细胞。与标准的CD3被破坏的异体CAR T细胞相比，CRAFT CAR T细胞在拥有更安全的基因组特征的同时，显示出同样强劲的细胞毒性。CRAFT CAR T细胞可作为双特异性T细胞衔接器（BiTE）的效应细胞，诱导对肿瘤细胞的CD3依赖性细胞毒性。值得注意的是，与CD3被破坏的CAR T细胞不同，CRAFT CAR T细胞存在于小鼠异种移植物中且无同种异体反应性。凭借MMEJ导向修复的启动和极小的脱靶，这种CRAFT crRNA可在临床环境中生成具有整体更低基因毒性特征的更安全的异体CAR T细胞。这一独特的CRAFT CAR T细胞群显示出体内持久性。此外，CRAFT CAR T可与BiTE疗法联合使用，为现有的体外异体CAR T细胞疗法提供了一种有前景且可能更持久的替代方案。
查看英文原文 English abstract
Several allogeneic CAR T-cell therapies are in clinical trials to evaluate safety, many of which rely on CRISPR/Cas-based genome editing. However, the Cas enzyme's random repair mechanism elevates the risk of undesired, off-target effects, posing a challenge for safe allogeneic CAR T-cell generation. To mitigate the risk of therapy-related oncogenesis, we sought to pursue an alternative safe strategy to manufacture allogeneic CAR T-cells distinct from the preexisting ones in the clinic. We have developed a CRISPR RNA (crRNA) targeting T-cell receptor beta constant (TRBC) gene, which in conjunction with AsCas12a Ultra enzyme, causes site-specific editing by eliciting a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, which mitigates off-targeting risk. Targeted amplicon sequencing was employed to evaluate off-targeting and repair mechanisms. Graft versus host alloreactivity as well as CAR T persistence was evaluated using immunocompromised mice. In vivo tumor challenge study was conducted for over 3 months. Engraftment confirmation was done using immunohistochemistry and flow cytometry. Interestingly while evaluating the edited T-cells, we sequestered a unique T-cell population that showed TCR disruption yet remained CD3-positive and did not cause in vivo alloreactivity. These CD3-Retained, Allogeneically Functioning T-cells (CRAFT-cells) showed a similar growth rate as unedited T-cells and were then used as a platform to produce CD19-targeted CAR T-cells and BAFF-R-targeted CAR T-cells. When compared to the standard CD3-disrupted allogeneic CAR T-cells, CRAFT CAR T-cells displayed an equally robust cytotoxicity while possessing a safer genomic profile. CRAFT CAR T-cells could function as effector cells for bispecific T-cell engager (BiTE) and induce CD3-dependent cytotoxicity against tumor cells. Pertinently, CRAFT CAR T-cells as opposed to CD3-disrupted CAR T-cells, were present in mouse xenografts without alloreactivity. Having the onset of MMEJ-directed repair and minimal off-targeting, positions this CRAFT crRNA to generate safer allogeneic CAR T-cells with an overall reduced genotoxic profile in a clinical setting. This unique CRAFT CAR T-cell population displays in vivo persistence. Additionally, CRAFT CAR T can be combined with BiTE therapy, offering a promising and potentially more durable alternative to preexisting ex vivo allogeneic CAR T-cell therapies.
利益披露 Disclosure
T. Hundal, None..
Y. Luo, None..
Y. Qie, None..
M. E. Gadd, None..
S. Guo, None..
M. A. Kharfan-Dabaja, None..
H. Qin, None.