New NK culture methods have emerged with a strong impact, assisting in the development of spot-type NK drugs

NK cells, as a type of innate lymphocytes that can non-specific kill tumor cells without antigen pre-sensitization, do not cause graft versus host disease, nor induce cytokine storms, making them less prone to long-term toxicity. This biological characteristic determines the high safety of NK cell therapy, which also indicates that NK cells have the potential to become universal ready-to-use drugs.

Although in recent years, companies worldwide have sprung up to launch NK cell therapies and a large amount of research on NK cells is also underway. Most NK cell therapies are still in the preclinical and clinical research stages，very few have entered the clinical phase I.

Map of global NK therapy trials as of May 8, 2023 (data source: Clinical trials. gov)

Ⅰ. Difficulties in NK cell culture

In NK cell therapy, the cultivation of NK cells is a crucial step. The following will introduce the difficulties of NK cell culture from the following aspects:

1. Difficulty in obtaining a sufficient number and high activity of NK cells

The number of NK cells in the human body is very small and their distribution is wide, so cultivating NK cells in vitro requires a lot of time and cost. In addition, due to the inherent characteristics of NK cells, such as sensitivity and stability, they are prone to inactivation or apoptosis during in vitro culture. Therefore, obtaining a sufficient number and high activity of NK cells is a challenge.

2. Difficulty in ensuring the purity of NK cells

During NK cell culture, there may be interference from other cell types, such as T cells, B cells, etc., which can affect the purity and activity of NK cells. Therefore, when cultivating NK cells, a series of effective measures need to be taken, such as cell sorting, medium selection, growth factor addition to ensure the purity and activity of NK cells.

3. Difficulty in controlling the proliferation and differentiation of NK cells

The proliferation and differentiation of NK cells are one of the most difficult stages to control in vitro culture. On the one hand, excessive proliferation can lead to cell apoptosis or differentiation into other types of cells, affecting the purity and activity of cells; On the other hand, excessive differentiation can lead to the loss of cell function and affect its application effectiveness. Therefore, it is necessary to control the proliferation and differentiation of NK cells through reasonable culture conditions and the addition of growth factors when cultivating them.

4. Difficulty in achieving large-scale production

At present, NK cell therapy is still in its early stages, and its clinical application still faces many challenges, including the challenge of achieving large-scale production. At the current technological level, producing high-quality NK cells requires a large amount of manpower, material resources, and financial resources, so how to achieve large-scale production is an important challenge.

Ⅱ. Current Status of NK Cell Culture

The outstanding effectiveness of NK cell therapy in treating malignant tumors is closely related to the limitations of existing culture methods, which have not been widely applied. It is difficult to culture and amplify NK cells in vitro. Regardless of the source of NK cells, peripheral blood, umbilical cord blood, or stem cell sources, amplification is currently a major bottleneck.

There are currently four commonly used methods for in vitro expansion of NK cells:

1. Trophoblast system

Generally, NK cells are induced and expanded by K562 cells or genetically modified K562 cells. The advantage of this method is that the cell expansion rate is high and the speed is fast, and it is easy to obtain high-phenotype CD56+ NK cells with high purity and strong lethality. However, the introduced trophoblast cells are tumor cells, and its potential safety risks are difficult to completely eliminate, which will also make it more difficult for NK cell products cultured in this way to be approved for drug application. In addition, normal cells and tumor cells are co-cultured, and such cells are used for treatment and reinfused into the body, which has great ethical risks and is difficult for patients to accept psychologically.

2. Peripheral blood or cord blood PBMC induction

Inducing PBMC to differentiate into NK cells by cytokines, this method uses factors that are originally present in the body, which is equivalent to simulating the in vivo environment and is safer. However, limited by the instability of the initial NK ratio of the blood sample, the final result of the culture may have large deviations, the production efficiency is relatively low, and a large number of cytokines are consumed during the culture process, which is costly. In addition, the level of pure factor culture technology on the market is uneven, which may lead to insufficient purity of cells, off-target effects, and weak killing effect.

Although there are many problems in the cultivation method of pure factors, its safety is high enough, which makes it less difficult for NK product declaration to be approved. Therefore, the pure factor culture method has become the current mainstream NK cell culture method. Moreover, with the advent of serum-free products and the iterative upgrade of NK cell culture reagents, more and more companies choose pure factor culture methods, which will further promote upstream companies to develop products with better performance and feedback downstream applications, thus forming a good market positive cycle.

3. NK-92 cell line

NK92 cells are an immortal cell line with high cytotoxicity and stable immune properties. NK92 cells are easier to obtain and expand, and easier to culture, but the shortcomings of NK92 are also obvious. It is too dependent on IL-2, which is a heavy burden on the human body, and lacks the expression of CD16, and has poor killing ability. It requires irradiation treatment, is tumorigenic, and has a very short survival time in the body.

4. NK-directed differentiation induced by iPSC or ESC

Inducing iPSCs or ESCs to differentiate into NK cells, this method has high technical barriers, the tumorigenicity of iPSCs has not been properly resolved, and the safety and clinical efficacy still need to face regulatory challenges. If it is to be applied in clinical practice, I am afraid there is still a long way to go.

To sum up, although there are many methods for culturing NK cells, they all have certain limitations, which is also a constraint on downstream applications. At present, everyone is generally optimistic about the pure factor culture method. The main consideration is that it is relatively safe, and safety is a special emphasis in current supervision. Drug declaration is an arduous and long process, and a good choice will save us from detours in this process.