來自胚胎干細胞的CAR-NK細胞抑制異種移植動物中人類b細胞惡性腫瘤的進展

2024年10月，中國科學院動物研究所干細胞與生殖生物學國家重點實驗室；北京干細胞與再生醫學研究所；暨南大學第一附屬醫院血液科(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老師研究團隊在《Cancer Immunity and Immunotherapy》上發表論文：

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

“來自胚胎干細胞的低免疫原性CD19 CAR-NK細胞抑制異種移植動物中人類b細胞惡性腫瘤的進展"

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：嵌合抗原受體（CAR）修飾的自然殺傷細胞（NK）具有不依賴mhc識別和強大的抗腫瘤功能等優勢。然而，來源于人體組織的同種異體CAR-NK細胞是異質的，容易被宿主清除。

方法：利用類器官聚集誘導法，制備B2M敲除、HLA-E和CD19 CAR異位表達的胚胎干細胞（ESC）系，使其正常分化并產生同質的CD19 CAR- nk （CD19 CAR- uink）細胞。將CD19 CAR-UiNK細胞與HLA錯配的外周血單個核細胞（PBMC）的T細胞或NK細胞共培養，評估CD19 CAR-UiNK細胞的免疫原性。該研究通過體外細胞毒性實驗和體內動物模型進一步評估了CD19 CAR-UiNK細胞對CD19+腫瘤細胞的治療作用。

結果：CD19 CAR-UiNK細胞表現出與未修飾NK細胞相似的活化和抑制受體以及NK細胞關鍵效應分子的典型表達模式。在共培養實驗中，CD19 CAR-UiNK細胞避開了同種異體T細胞的反應，抑制了同種異體NK細胞的反應。在功能上，CD19 CAR-UiNK細胞強烈分泌IFN-γ和TNF-α，并在Nalm-6腫瘤細胞刺激下上調CD107a。CD19 CAR-UiNK細胞在體外有效地清除了CD19+腫瘤細胞，包括b細胞癌細胞系和來自人b細胞白血病和淋巴瘤的原代腫瘤細胞。此外，CD19 CAR-UiNK細胞在異種移植動物中表現出很強的抗腫瘤活性。

結論：該研究提供了一種從ESCs中獲得具有強大抗腫瘤作用的均勻性和低免疫原性CD19 CAR-iNK細胞的策略。該研究的研究對開發低免疫原性CD19 CAR-NK細胞療法具有重要意義，該療法使用人類ESC作為無限細胞來源。

該論文中，ESC、OP9細胞、表達熒光素酶的Nalm-6細胞、從三名患者的骨髓中分離出原發性人類白血病和淋巴瘤細胞的體外培養是使用Ausbian特級胎牛血清完成的。