肿瘤细胞治疗研究
英文原题：HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity.
我们已在临床前验证了一种 iCAR NOT 门控技术，该技术广泛适用于在 A2 LOH 背景下靶向 HER2 表达。
背景：实体瘤CAR-T细胞疗法的一项主要挑战，是重要组织和器官表达CAR靶向肿瘤抗原所导致的靶向肿瘤外毒性。我们描述一种双CAR NOT逻辑门，其中抑制性CAR（iCAR）识别HLA-A*02（“A2”）；当肿瘤发生A2杂合性缺失（LOH）时，该设计可利用强效HER2激活性CAR（aCAR）实现有效治疗。 方法：开展CAR-T细胞筛选，鉴定可用于NOT逻辑门的天然免疫受体抑制性结构域（iDomain），使其能够高特异性杀伤A2−HER2+肺癌细胞系，同时保护A2+HER2+肺癌细胞系。对领先候选物进行广泛分析，包括T细胞活化和杀伤、连续挑战中的可逆性和持久性、混合3D球体及2D培养中的靶细胞特异性，以及CAR表达水平和细胞迁移特征。 结果：鉴定出白细胞免疫球蛋白样受体B1（LIR1）iDomain iCAR，在调节第二代HER2 aCAR细胞毒性方面效果最佳。靶点转移实验显示，LIR1 NOT门CAR-T细胞的“开”和“关”细胞状态既持久又可逆。保护作用需要iCAR信号传导，并与aCAR和iCAR表面表达降低有关。iCAR调控足以在3D邻接球体检测中实现高度靶向特异性；该检测用于模拟克隆性A2 LOH病灶与正常组织的交界处。然而，在混合培养中，我们观察到明显的对A2+细胞旁观者杀伤，机制依赖且不依赖aCAR。在体内，LIR1 NOT门CAR-T细胞可保护免受H1703-A2+肿瘤侵袭，并对H1703-A2−肿瘤发挥高效作用。我们观察到，A2+供者中的iCAR因顺式结合而失活，但敲除HLA-A可完全恢复iCAR活性。 结论：我们在临床前验证了一种iCAR NOT门技术，可广泛用于A2 LOH背景下靶向HER2表达。该策略旨在避免肿瘤外毒性，同时维持高效抗肿瘤活性。
BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2 - HER2 + lung cancer cell lines but spare A2 + HER2 + lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2 + cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2 + tumors and high efficacy against H1703-A2 - tumors in-vivo. We observed that the iCAR is inactive in A2 + donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.
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