来自胚胎干细胞的CAR-NK细胞抑制异种移植动物中人类b细胞恶性肿瘤的进展

2024年10月，中国科学院动物研究所干细胞与生殖生物学国家重点实验室；北京干细胞与再生医学研究所；暨南大学第一附属医院血液科(State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China；Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China；Department of Hematology, The First Affiliated Hospital of Jinan University, Guangzhou, China) Qi Zhang老师研究团队在《Cancer Immunity and Immunotherapy》上发表论文：

“Hypoimmunogenic CD19 CAR-NK cells derived from embryonic stem cells suppress the progression of human B-cell malignancies in xenograft animals"

“来自胚胎干细胞的低免疫原性CD19 CAR-NK细胞抑制异种移植动物中人类b细胞恶性肿瘤的进展"

Abstract：

Background: Chimeric antigen receptor (CAR) engineered natural killer (NK) cells exhibit advantages such as MHC-independent recognition and strong anti-tumor functions. However, allogeneic CAR-NK cells derived from human tissues are heterogeneous and susceptible to clearance by hosts.

Methods: We generated a B2M knockout, HLA-E and CD19 CAR ectopic expressing embryonic stem cell (ESC) line, which differentiated normally and gave rise to homogeneous CD19 CAR-NK (CD19 CAR-UiNK) cells using an organoid aggregate induction method. The CD19 CAR-UiNK were co-cultured with T cells or NK cells derived from peripheral blood mononuclear cells (PBMC) with the mismatched HLA to evaluate the immunogenicity of CD19 CAR-UiNK cells. We further assessed the therapeutic effects of CD19 CAR-UiNK cells on CD19⁺ tumor cells through in vitro cytotoxicity assays and in vivo animal models.

Results: The CD19 CAR-UiNK cells exhibited typical expression patterns of activating and inhibitory receptors, and crucial effector molecules of NK cells, similar to those of unmodified NK cells. In co-culture assays, the CD19 CAR-UiNK cells evaded allogeneic T cell response and suppressed allogeneic NK cell response. Functionally, the CD19 CAR-UiNK cells robustly secreted IFN-γ and TNF-α, and upregulated CD107a upon stimulation with Nalm-6 tumor cells. The CD19 CAR-UiNK cells effectively eliminated CD19⁺ tumor cells in vitro, including B-cell cancer cell lines and primary tumor cells from human B-cell leukemia and lymphoma. Further, the CD19 CAR-UiNK cells exhibited strong anti-tumor activity in xenograft animals.

Conclusion: We offer a strategy for deriving homogeneous and hypoimmunogenic CD19 CAR-iNK cells with robust anti-tumor effects from ESCs. Our study has significant implications for developing hypoimmunogenic CD19 CAR-NK cell therapy using human ESC as an unlimited cell source.

摘要：

背景：嵌合抗原受体（CAR）修饰的自然杀伤细胞（NK）具有不依赖mhc识别和强大的抗肿瘤功能等优势。然而，来源于人体组织的同种异体CAR-NK细胞是异质的，容易被宿主清除。

方法：利用类器官聚集诱导法，制备B2M敲除、HLA-E和CD19 CAR异位表达的胚胎干细胞（ESC）系，使其正常分化并产生同质的CD19 CAR- nk （CD19 CAR- uink）细胞。将CD19 CAR-UiNK细胞与HLA错配的外周血单个核细胞（PBMC）的T细胞或NK细胞共培养，评估CD19 CAR-UiNK细胞的免疫原性。该研究通过体外细胞毒性实验和体内动物模型进一步评估了CD19 CAR-UiNK细胞对CD19+肿瘤细胞的治疗作用。

结果：CD19 CAR-UiNK细胞表现出与未修饰NK细胞相似的活化和抑制受体以及NK细胞关键效应分子的典型表达模式。在共培养实验中，CD19 CAR-UiNK细胞避开了同种异体T细胞的反应，抑制了同种异体NK细胞的反应。在功能上，CD19 CAR-UiNK细胞强烈分泌IFN-γ和TNF-α，并在Nalm-6肿瘤细胞刺激下上调CD107a。CD19 CAR-UiNK细胞在体外有效地清除了CD19+肿瘤细胞，包括b细胞癌细胞系和来自人b细胞白血病和淋巴瘤的原代肿瘤细胞。此外，CD19 CAR-UiNK细胞在异种移植动物中表现出很强的抗肿瘤活性。

结论：该研究提供了一种从ESCs中获得具有强大抗肿瘤作用的均匀性和低免疫原性CD19 CAR-iNK细胞的策略。该研究的研究对开发低免疫原性CD19 CAR-NK细胞疗法具有重要意义，该疗法使用人类ESC作为无限细胞来源。

该论文中，ESC、OP9细胞、表达荧光素酶的Nalm-6细胞、从三名患者的骨髓中分离出原发性人类白血病和淋巴瘤细胞的体外培养是使用Ausbian特级胎牛血清完成的。