肿瘤细胞治疗研究
英文原题：Loop33 × 123 CAR-T targeting CD33 and CD123 against immune escape in acute myeloid leukemia.
靶向 CD33 和 CD123 的 Loop33 123 CAR-T 能够高效清除 AML 细胞并延长荷瘤小鼠的生存期，同时解决免疫逃逸问题。
背景：过去十年中，靶向CD33或CD123的CAR-T（CAR-T）细胞等免疫疗法已得到充分开发，用于治疗急性髓系白血病（AML）。然而，无法维持无肿瘤生存以及抗原丢失导致复发的可能性令人担忧。为改善结局，需要同时靶向CD33和CD123。 方法：基于我们此前构建的CD33和CD123单价CAR-T细胞，我们采用分子克隆技术构建Loop33-123和Loop123-33 CAR-T细胞。所有CAR-T细胞均通过慢病毒转导健康供者T细胞制备。第7天检测细胞活化、耗竭及亚型比例，以评估表型。采用多种AML细胞系和原代AML细胞进行共培养杀伤实验，并通过流式细胞术检测脱颗粒和细胞因子分泌水平。采用野生型Molm 13细胞系，或Molm 13-KO33与Molm 13-KO123细胞混合物，建立细胞来源异种移植模型，以模拟免疫逃逸。通过监测荷瘤小鼠体重和生存，进一步评估Loop33-123和Loop123-33 CAR-T细胞的体内疗效。 结果：体外研究显示，Loop33-123 CAR-T细胞可有效清除AML细胞系和原代AML细胞，并增强脱颗粒和细胞因子分泌。与我们此前构建的CD33或CD123单价CAR-T细胞相比，Loop33-123 CAR-T细胞在免疫逃逸模型中优势更明显。体内研究进一步证实，Loop33-123 CAR-T细胞可有效延长小鼠生存，且无明显毒性。然而，Loop123-33 CAR-T细胞未显示相同效果。此外，Loop33-123 CAR-T细胞可有效规避免疫逃逸，而单价CAR-T细胞无法做到这一点。 结论：靶向CD33和CD123的Loop33-123 CAR-T细胞可有效清除AML细胞并延长荷瘤小鼠生存，同时应对免疫逃逸问题。
BACKGROUND: Immunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes. METHODS: Based on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 123 and Loop123 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 123 and Loop123 33 CAR-T cells were further assessed for their efficacy in vivo. RESULTS: In vitro study, our results demonstrated that Loop33 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed. CONCLUSIONS: Loop33 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape.
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