肿瘤细胞治疗研究
英文原题：Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors.
我们这种新型 CAR-NK 策略利用了 HLA-G 兼具肿瘤相关新抗原和免疫检查点（ICP）的双重性质，以对抗肿瘤扩散。
背景：实体瘤免疫治疗长期受阻于免疫抑制性肿瘤微环境的形成，以及缺乏可用于不同实体瘤的特异性肿瘤相关抗原。人白细胞抗原G（HLA-G）是一种免疫检查点蛋白（ICP），多数肿瘤细胞会新表达该蛋白以逃避免疫攻击；近期体外研究已证实其可作为白血病嵌合抗原受体（CAR）T细胞治疗的有效靶点。我们在此设计并测试利用CAR策略靶向实体瘤中的HLA-G。方法：我们开发了一种新型CAR策略，以自然杀伤（NK）细胞为效应细胞，并利用DAP12介导的胞内信号放大增强细胞毒作用。以抗HLA-G单链可变片段（scFv）作为靶向结构域，并在4种不同实体瘤模型中进行体内外测试。我们还评估低剂量化疗与抗HLA-G CAR-NK策略联合治疗的协同作用。结果：转导HLA-G CAR的NK细胞可有效体外杀伤乳腺癌、脑癌、胰腺癌和卵巢癌细胞；在原位小鼠模型中，该疗法减少异种移植瘤生长并延长中位生存期。在肿瘤共培养实验中，抗HLA-G scFv结构域促进NK细胞Syk/Zap70激活，提示其可能逆转HLA-G介导的免疫抑制，从而恢复NK细胞固有的细胞毒功能。低剂量化疗可进一步诱导肿瘤HLA-G表达；联合抗HLA-G CAR-NK后，体内外均观察到广泛肿瘤消融。肿瘤HLA-G上调涉及DNMT1抑制及抗原加工相关转运蛋白1（TAP1）启动子去甲基化。结论：我们的新型CAR-NK策略利用HLA-G同时作为肿瘤相关新抗原和ICP的双重特性，以抑制肿瘤扩散。临床常用化疗药物联合给药可进一步增强肿瘤消融。该新策略已具备进一步开发条件，有望广泛用于实体瘤治疗。
BACKGROUND: Immunotherapy against solid tumors has long been hampered by the development of immunosuppressive tumor microenvironment, and the lack of a specific tumor-associated antigen that could be targeted in different kinds of solid tumors. Human leukocyte antigen G (HLA-G) is an immune checkpoint protein (ICP) that is neoexpressed in most tumor cells as a way to evade immune attack and has been recently demonstrated as a useful target for chimeric antigen receptor (CAR)-T therapy of leukemia by in vitro studies. Here, we design and test for targeting HLA-G in solid tumors using a CAR strategy. METHODS: We developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification. A single-chain variable fragment (scFv) against HLA-G is designed as the targeting moiety, and the construct is tested both in vitro and in vivo on four different solid tumor models. We also evaluated the synergy of this anti-HLA-G CAR-NK strategy with low-dose chemotherapy as combination therapy. RESULTS: HLA-G CAR-transduced NK cells present effective cytolysis of breast, brain, pancreatic, and ovarian cancer cells in vitro, as well as reduced xenograft tumor growth with extended median survival in orthotopic mouse models. In tumor coculture assays, the anti-HLA-G scFv moiety promotes Syk/Zap70 activation of NK cells, suggesting reversal of the HLA-G-mediated immunosuppression and hence restoration of native NK cytolytic functions. Tumor expression of HLA-G can be further induced using low-dose chemotherapy, which when combined with anti-HLA-G CAR-NK results in extensive tumor ablation both in vitro and in vivo. This upregulation of tumor HLA-G involves inhibition of DNMT1 and demethylation of transporter associated with antigen processing 1 promoter. CONCLUSIONS: Our novel CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread. Further ablation of tumors can be boosted when combined with administration of chemotherapeutic agents in clinical use. The readiness of this novel strategy envisions a wide applicability in treating solid tumors.
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