citations

                    Robust differentiation of NK cells from MSLN.CAR-IL-15–engineered human iPSCs with enhanced antitumor efficacy against solid tumors
                

Authors:

Jiang Q, Yu W, Ma J, Zhao M, Zou J, Mir S, Zhang J, Germain RN, Hassan R.

In:

Source: Sci Adv.
Publication Date: (2025)
Issue: 11(18): eadt9932

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Stem Cells

Gene Expression

Basic Research

Regenerative medicine

Drug Discovery

Cells used in publication:

Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Platform:

4D-Nucleofector® X-Unit

Experiment

Stable CAR constructs expression in the LiPSC-GR1.1 line
LiPSC-GR1.1 cells were dissociated as a single-cell suspension using TrypLE and then washed with fresh E8 medium supplemented with ROCKi. A total of 0.2 million iPSCs was harvested through centrifugation, resuspended in 20 µl of P3 primary solution containing 1 µg of CAR-expressing PBCAG transposon vector plus 0.25 µg of Super PiggyBac Transposase (cat. no. PB210PA-1, System Bioscience), and transferred into nucleocuvette strips. Nucleofection was performed using Lonza 4D Nucleofector with pulse setting CA-137 according to the manufacturer’s guidelines. The iPSCs nucleofected without PBCAG transposon were used as mock control. CAR-expressing iPSCs were enriched through FACS of EGFR+ iPSCs during days 7 to 9 postnucleofection.

Abstract

Human induced pluripotent stem cells (iPSCs) offer a promising source for chimeric antigen receptor (CAR)–engineered natural killer (NK) products. However, complex iPSC-NK (iNK) manufacturing challenges clinical use. Here, we identified LiPSC-GR1.1 as a superior iPSC line for iNK production. By engineering LiPSC-GR1.1 with a mesothelin (MSLN)–targeting CAR and interleukin-15 (IL-15), we achieved robust differentiation of iPSCs into mature activated iNK cells with enhanced tumor killing efficacy, superior tumor homing, and vigorous proliferation. Single-cell transcriptomic analysis revealed that transforming growth factor–ß (TGF-ß)– producing tumor cells up-regulated major histocompatibility complex molecules and down-regulated MSLN post–CAR-IL- 15 iNK treatment. Tumor-infiltrating CAR-IL- 15 iNK cells exhibited high levels of CAR, IL-15, and NK-activating receptors, negligible checkpoint exhaustion markers, and extremely low levels of NK suppressive factors CISH, TGFBR2, and BATF, enabling them to sustain activation, metabolic fitness, and effective tumor killing within TGF-ß– rich hypoxic tumor microenvironment. Overall, we developed MSLN.CAR-IL-
15– engineered GR1.1-iNK therapy with enhanced antitumor efficacy for solid tumor treatment.

Open in PubMed