肿瘤细胞治疗研究
英文原题：CD33(KO)-CD33-mesothelin Loop CAR design avoids fratricide and improves efficacy of iNK cells against acute myeloid leukemia.
CD33(KO)-CD33-mesothelin Loop CAR design avoids fratricide and improves efficacy of iNK cells against acute myeloid leukemia.
Loop CAR 赋予 UCB-NK 细胞和 hPSC-iNK 细胞更强的针对 CD33 + MSLN + 肿瘤细胞的细胞毒性。
背景：急性髓系白血病（AML）患者常为老年人，自体嵌合抗原受体（CAR）T细胞疗法的耐受性及持久疗效面临挑战。异基因CAR自然杀伤（NK）细胞疗法可能减轻毒性，并增强针对AML的抗白血病潜力。靶向CD33的CAR-NK细胞已用于AML治疗研究，但CD33 CAR-NK细胞会杀伤表达CD33的NK细胞，这种自相杀伤限制了其扩增和疗效。间皮素（MSLN）是一种肿瘤分化抗原，在部分AML患者中高表达，因此是有前景的AML治疗靶点。 方法：我们设计了一种新型CD33-MSLN环形CAR（Loop CAR），并在脐带血来源人NK（UCB-NK）细胞和人多能干细胞来源NK（hPSC-iNK）细胞中评估其抗肿瘤疗效。为进一步避免NK细胞内源性CD33表达导致的自相杀伤，我们通过敲除CD33基因建立hPSC来源细胞系，并工程化构建Loop CAR。采用类器官诱导方法制备CD33敲除Loop CAR-iNK细胞。在体外及AML异种移植小鼠中，研究CD33敲除Loop CAR-iNK细胞针对表达CD33和MSLN的肿瘤细胞的疗效。 结果：与CD33 CAR-NK和MSLN CAR-NK细胞相比，Loop CAR-NK细胞对双抗原阳性肿瘤细胞系和原发AML细胞具有更强细胞毒性。此外，Loop CAR-NK细胞中与NK细胞活化和细胞毒功能相关的信号通路上调。在iNK细胞中敲除CD33有效避免了自相杀伤、改善扩增能力，并显著增强Loop CAR-iNK细胞经CD33和MSLN介导的特异性细胞毒性。此外，CD33敲除Loop CAR-iNK细胞在AML异种移植小鼠模型中具有更强肿瘤杀伤活性，并显著延长小鼠生存期。 结论：Loop CAR使UCB-NK和hPSC-iNK细胞对CD33+ MSLN+肿瘤细胞产生更强细胞毒性。遗传性破坏CD33可避免自相杀伤并提高Loop CAR-iNK细胞治疗AML的疗效。这一创新策略具有独特优势和转化潜力。
BACKGROUND: Patients with acute myeloid leukemia (AML) are often older, which brings challenges of endurance and persistent efficacy of autologous chimeric antigen receptor (CAR)-T cell therapies. Allogenic CAR-natural killer (NK) cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. CD33 CAR-NK cells have been investigated for AML therapy. However, the fratricide-mediated lysis of CD33-expressing NK cells by CD33 CAR-NK cells limits the expansion and efficacy of CD33 CAR-NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is highly expressed in a fraction of patients with AML, making it a promising target for AML therapy. METHODS: We designed a novel CD33-MSLN Loop CAR (Loop CAR) and evaluated its antitumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established an hPSC-derived cell line via knockout of the CD33 gene (CD33 KO ) and engineered Loop CAR. We generated CD33 KO -Loop CAR-iNK cells using an organoid induction approach. The efficacy of CD33 KO -Loop CAR-iNK cells against tumor cells expressing CD33 and MSLN was investigated both in vitro and in AML xenograft mice. RESULTS: Loop CAR-NK cells exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells compared with CD33 CAR-NK and MSLN CAR-NK cells. Moreover, Loop CAR-NK cells showed upregulated signaling pathways related to NK cell activation and cytotoxic function. The loss of CD33 in iNK cells effectively avoided fratricide, improved expansion ability, and significantly enhanced CD33 and MSLN-mediated specific cytotoxicity of Loop CAR-iNK cells. Moreover, the CD33 KO -Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mouse models and significantly prolonged mouse survival. CONCLUSION: Loop CAR empowered both UCB-NK cells and hPSC-iNK cells with superior cytotoxicity against CD33 + MSLN + tumor cells. Genetic disruption of CD33 avoided fratricide and improved efficacy of Loop CAR-iNK cells against AML. This innovative strategy possesses unique advantages and translational potential for treating AML.
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