image: Overview of the method for large-scale production of iNK and CAR-iNK cells from cord blood CD34+ hematopoietic stem and progenitor cells.
Credit: Image by Prof. WANG Jinyong's Lab
Chinese researchers have developed a novel method to efficiently engineer natural killer (NK) cells for cancer immunotherapy.
NK cells are central to early antiviral and anticancer defense—among other immune system roles—making them well-suited for cancer immunotherapy. For example, chimeric antigen receptor (CAR)-NK therapy involves adding a lab-built receptor (a CAR) to an NK cell, enabling it to recognize a specific antigen on a cancer cell and attack it.
However, conventional CAR-NK immunotherapies rely primarily on mature NK cells isolated from human tissues, such as peripheral blood or cord blood, which poses multiple challenges, including high heterogeneity, low engineering efficiency, high handling costs, and time-intensive processing.
Now a research team led by Prof. WANG Jinyong from the Institute of Zoology of the Chinese Academy of Sciences has developed a novel method to generate induced (that is, lab-generated) NK (iNK) cells and CAR-engineered iNK (CAR-iNK) cells from CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood.
The study was recently published in Nature Biomedical Engineering.
Past attempts to generate NK cells from cord blood-derived CD34+ HSPCs have been hindered by poor induction efficiency and the resulting cells' functional immaturity. To overcome these barriers, the researchers shifted the genetic engineering to the earlier CD34+ HSPC stage, combining CAR transduction, efficient progenitor expansion, and NK-lineage commitment.
The three-step process began with expanding CD34+ HSPCs (or CD19 CAR-transduced HSPCs) using irradiated AFT024 feeder cells, achieving approximately 800- to 1,000-fold growth within 14 days. Next, the expanded cells were co-cultured with OP9 feeder cells to form artificial hematopoietic organoid aggregates—structures that promote efficient NK lineage commitment and development. Finally, the cells committed to the NK lineage underwent maturation and proliferation, yielding highly pure iNK or CAR-iNK cells that expressed endogenous CD16.
Notably, the method demonstrated that a single CD34+ HSPC could produce up to 14 million iNK cells or 7.6 million CAR-iNK cells. In theory, just one-fifth of a standard cord blood unit could generate enough cells to supply thousands—or even tens of thousands—of therapeutic doses.
A major advance is the drastic reduction in the amount of viral vector required for CAR engineering. Compared with the viral load typically needed to engineer mature NK cells, the new method used only ~1/140,000 (by Day 42 of culture) to ~1/600,000 (by Day 49) as much vector.
In functional tests, the team confirmed that both iNK and CAR-iNK cells exhibited strong tumor-killing activity. In cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of human B-cell acute lymphoblastic leukemia (B-ALL), CD19 CAR-iNK cells suppressed tumor growth and prolonged the survival of the animals.
The new method not only boosted the induction efficiency of iNK and CAR-iNK cells but also substantially lowered the cost of CAR engineering, the researchers noted.
Funding for the research was provided by the Ministry of Science and Technology of the People's Republic of China and the National Natural Science Foundation of China, among others.
Journal
Nature Biomedical Engineering