携带CCL19、CCR2B、高亲和力CD16、IL-15和NKG2D复合物的人诱导多能干细胞衍生的自然杀伤细胞增强抗实体瘤活性。

Human iPSC-derived NK cells armed with CCL19, CCR2B, high-affinity CD16, IL-15, and NKG2D complex enhance anti-solid tumor activity.

作者信息

Fukutani Yuma, Kurachi Kenji, Torisawa Yu-Suke, Miyata Kotoko, Hayashi Makoto, Sasaki Kaoru, Saitoh Kodai, Watanabe Sono, Hasegawa Yudai, Naritomi Yoichi, Igarashi Yuka, Goto Kumiko, Sato Yuka, Uesugi Noriko, Murai Hidetaka, Sakurai Tetsuya, Ozaki Toru, Tsuneyoshi Norihiro, Yamada Masashi, Takeno Yuriko, Hosoya Tomonori, Nishigaki Fusako, Kimura Hironobu, Tamura Kouichi

机构信息

Research Division, Kobe Research Institute, HEALIOS K.K., Kobe KIMEC Center Bldg. 3F, 1-5-2 Minatojima-Minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan.

出版信息

Stem Cell Res Ther. 2025 Jul 15;16(1):373. doi: 10.1186/s13287-025-04461-9.

DOI:10.1186/s13287-025-04461-9

PMID:40660312

Abstract

BACKGROUND

Studies of chimeric antigen receptor (CAR)-T and -Natural killer (NK) cells have shown promising results in treating hematological malignancies. However, there are still obstacles to effectively treating solid tumors. These include the challenges of CAR-T cell homing and infiltration, the presence of immunosuppressive microenvironments, and the potential for antigen escape in solid tumors. To overcome the known limitations of immune cell therapy, we engineered human induced pluripotent stem cell (hiPSC)-derived NK cells armed with CCL19, CCR2B, high-affinity CD16, IL-15, and NKG2D-DAP10 complex.

METHODS

We introduced the six genes, CCL19, CCR2B, FCGR3A (CD16), IL-15, KLRK1 (NKG2D), and HCST (DAP10), which were controlled under human EF1a promoter, into hiPSCs using the piggyBac system and differentiated them into NK cells. We evaluate the antitumor function, including killing activity, antibody-dependent cytotoxicity, migration ability, and recruitment of dendritic cells. In addition, in vivo antitumor activity was determined by using an orthotopic lung cancer mouse model.

RESULTS

The gene-engineered hiPSCs expressed all six transgenes, showed normal karyotypes, and were able to differentiate into CD56 NK cells. The gene-engineered hiPSC-derived NK (eNK) cells showed improvement in viability without additional cytokine supplement in vitro and in vivo. Overexpression of NKG2D complex and high-affinity CD16 enhanced the antitumor function of the eNK cells. Forced expression of CCR2B enhanced eNK cell tumor infiltration. Forced expression of CCL19 endowed the eNK cells with the ability to recruit dendritic cells. We found that the eNK cells were able to lyse HLA-E-expressing tumor cells, but not normal human cells. Moreover, eNK cells demonstrated superior anti-tumor activity in an orthotropic lung cancer mouse model.

CONCLUSION

These proof-of-concept studies demonstrate the promise of our eNK cells as a novel adoptive cell therapy product for the treatment of solid tumors.

摘要

背景

嵌合抗原受体（CAR）-T细胞和自然杀伤（NK）细胞的研究在治疗血液系统恶性肿瘤方面已显示出有前景的结果。然而，在有效治疗实体瘤方面仍存在障碍。这些障碍包括CAR-T细胞归巢和浸润的挑战、免疫抑制微环境的存在以及实体瘤中抗原逃逸的可能性。为了克服免疫细胞疗法的已知局限性，我们构建了携带CCL19、CCR2B、高亲和力CD16、IL-15和NKG2D-DAP10复合物的人诱导多能干细胞（hiPSC）衍生的NK细胞。

方法

我们使用piggyBac系统将受人类EF1a启动子控制的六个基因CCL19、CCR2B、FCGR3A（CD16）、IL-15、KLRK1（NKG2D）和HCST（DAP10）导入hiPSC，并将它们分化为NK细胞。我们评估了其抗肿瘤功能，包括杀伤活性、抗体依赖性细胞毒性、迁移能力和树突状细胞的募集。此外，通过使用原位肺癌小鼠模型确定体内抗肿瘤活性。

结果

基因工程改造的hiPSC表达所有六个转基因，显示正常核型，并能够分化为CD56 NK细胞。基因工程改造的hiPSC衍生的NK（eNK）细胞在体外和体内无需额外添加细胞因子的情况下，活力有所提高。NKG2D复合物和高亲和力CD16的过表达增强了eNK细胞的抗肿瘤功能。CCR2B的强制表达增强了eNK细胞的肿瘤浸润。CCL19的强制表达赋予eNK细胞募集树突状细胞的能力。我们发现eNK细胞能够裂解表达HLA-E的肿瘤细胞，但不能裂解正常人类细胞。此外，eNK细胞在原位肺癌小鼠模型中表现出卓越的抗肿瘤活性。