CAR NK: A New Era in Cancer Treatment with Natural Killer Immune Cells

Created by modifying T cells, CAR T treatments first received approval for leukemia and lymphoma in 2017 from the United States Food and Drug Administration (FDA), beginning a new era of cellular immunotherapy in cancer treatment. We now face a new living cancer medicine with greater efficacy and fewer side effects: CAR NK cell therapy.

The immune system’s “natural killer” (narutal killer = NK) cells belong to the innate immune system and were first discovered in mice in 1975. With different cell-surface receptors, NK cells have different distribution patterns across healthy tissues, but most are found in the blood, liver, spleen and bone marrow. A small proportion is also found in lymph codes. NK cells are on the front line of protecting the body against pathogens. When normal body cells are invaded by a virus, they rapidly become active and generate protective responses without requiring a previous encounter.

Advances in technology and studies in recent years have helped us better understand tumor immunology and its relationship with the immune system. Research on NK cells is of great interest to investigators. NK cells obtained from various sources, such as a donor’s bone marrow, human embryonic stem cells, induced pluripotent stem cells, peripheral blood and umbilical cord blood, and transferred to a patient were found to generate stronger responses against tumors. However, isolating these NK cells and expanding them in the laboratory is extremely difficult. Cell-cloning technology has enabled the creation of highly pure, functionally consistent NK cell lines with the same characteristics: KHYG-1, NK-92, NKL, NKG and YT. These lines have made large-scale experiments easier. Modification of the NK-92 cell line with CAR (chimeric antigen receptor) produced strong antitumor properties and paved the way for clinical use.

Although NK cells display various biomarkers on their surface, the cell-surface markers CD56 and CD16 are the most notable. Depending on the intensity of CD56 and CD16 expression, NK cells are divided into two groups: CD56bright and CD56dim.

CD56dim NK cells are fully mature and account for approximately 90% of NK cells in the blood. This group is responsible for cytotoxic/cell-killing responses and plays a role in antibody-dependent cell-mediated cytotoxicity. By contrast, CD56bright NK cells are immature and are mainly found in tissues and secondary lymphoid organs. CD56bright cells display very little CD16 on their surface. Their main role in the immune response is therefore to secrete cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, granulocyte-macrophage colony-stimulating (GMCS) factor, IL-10 and IL-13.

NK cells recognize ‘foreign’ cells with the help of the ‘activating NK receptors’ and ‘inhibitory NK receptors’ displayed on their surface. These receptors recognize the major histocompatibility complex I (MHC-I) molecule found on the surface of all nucleated cells in the body. This indicates that the opposing cell is healthy, activates the inhibitory NK receptor, and prevents NK cells from destroying the target.

Explanation of the figure above: a) Self / friendly cell: The NK cell recognizes the MHC-I molecule on the cell surface and refrains from destroying the target cell. b) Missing-self cell: The target cell does not display the MHC-I molecule on its surface. In this situation, the NK cell receives stimulatory signals through its activating receptors and begins to destroy the cell. c) Stressed cell: When the target cell displays altered MHC-I or stress-induced molecules on its surface, it is recognized and destroyed by NK cells. d) Non-self cell: The target cell displays foreign MHC-I on its surface and stimulates the cytotoxic mechanisms of NK cells. The target cell is thus destroyed.

When healthy cells are invaded by a virus or a tumor develops, the MHC-I molecule displayed on the surface disappears. This activates the activating NK cell receptor, which destroys the target cell through three different pathways:

1. Perforin/Granzyme pathway: Perforin stored in vesicles inside NK cells is released outside the cell and increases permeability at the target cell surface. Granzyme is then delivered, causing apoptosis and leading to cell death.
2. Fas/FasL pathway: When two molecules called Fas and FasL bind together, a death signal is sent to the target cell, which is destroyed by apoptosis within a few hours.
3. Cytokine pathway: Cytokines secreted by NK cells, such as TNF-alpha, disrupt lysosomal stability in the target cell. This releases hydrolytic enzymes that break down the target cell’s DNA, destroying the cell.

NK-92 cells

The NK-92 cell line is the only NK cell line approved by the United States Food and Drug Administration (FDA) for use in phase 1 and phase 2 clinical studies. NK-92 has almost no ‘’inhibitory receptors’ but displays abundant ‘’activating’ receptors. Its high levels of perforin and granzyme also indicate broad cytotoxic activity.

Many studies using the NK-92 cell line have evaluated its efficacy in cancer treatment. For example, studies using leukemia and nyelom cells found that NK-92 cells had a strong cytotoxic effect and effectively destroyed them.

Chimeric antigen receptors (CAR) and the NK-92 cell line

Although CD19-targeted CAR T cell therapy has received FDA approval, the method has many limitations, particularly unwanted side effects and cytokine storms. CAR T cell therapy does not produce successful results in solid tumors, and production using T cells obtained from the patient can take a long time and involve difficulties collecting enough cells. Although obtaining T cells from a donor could overcome these problems, it also carries a risk of graft-versus-host disease, which causes organ dysfunction.

The high cytotoxic activity of CAR NK-92 cells and the ability to produce large quantities of homogeneous cells quickly are among their greatest advantages.

Their shorter lifespan in the body compared with CAR-T cells also reduces potential side effects.

The mechanism of action of anti-CD19 CAR-NK therapy is summarized simply below.

What Are the Advantages of CAR NK-92 Cell Therapy?

CAR NK-92 cell therapy rivals CAR T cell therapy because of its many advantages. These include;

1. CAR NK-92 cell therapy does not cause graft-versus-host disease and has more effective cytotoxic activity.
2. CAR NK-92 cells can target the tumor directly and destroy the target cell through NK-92 cell activation.
3. Even if the targeted antigen disappears from the tumor surface, CAR NK-92 cells remain active through their ‘activating receptors’.
4. ‘Inhibitory receptors’ are expressed at low levels on the surface of NK-92 cells. Removing the existing receptors as well makes NK-92 cells more resilient against solid tumors than other immune cells.
5. Immortal NK-92 cells have a stable phenotype/appearance and can be expanded in the laboratory for a long time.
6. The T cells needed for CAR T cell therapy must be collected from the patient’s own blood. However, problems include insufficient numbers of T cells or abnormalities in the available cells that make them unable to recognize tumor cells adequately. A person can also produce only 1 dose of CAR-T at a time, which increases costs. There is no such limitation for NK cells. NK cells collected from a donor can also be used for CAR-NK therapy.

Below are cancer clinical trials in which CAR NK cell therapy has been tested:

What Are the Challenges of CAR NK-92 Cell Therapy?

Despite their effective antitumor properties, problems remain to be resolved:

• The NK-92 cell line was obtained from patients with malignant non-Hodgkin lymphoma. This may cause a secondary tumor to develop. It may also cause susceptibility to Epstein-Barr virus following NK-92 cell transfer. To improve safety, the cells should therefore be irradiated before clinical use to prevent potential side effects. Studies have shown that even after irradiation, NK-92 cells retain their ability to proliferate and kill.
• Exposure to high doses of radiation shortens the half-life of NK-92 cells, which also reduces their antitumor properties. Multiple CAR NK-92 injections may be needed to prevent this.
• The most common method of initiating CAR expression in NK cells is genetic modification using viral vectors. Different vectors may be used for this purpose. The aim is to introduce the genetic information needed for CAR expression into the NK cell. However, this method can also have disadvantages. For example, the vectors used can be costly and have long, complex production cycles. There is also no guarantee that the vectors will insert the intended genetic information into the desired location. This may damage normal genes and initiate abnormal cell cycles. The CRISPR-Cas9 system is a method introduced in recent years to address these problems.
• CAR NK-92 cells attack by targeting a specific antigen on the tumor. However, unwanted toxic effects may occur if these antigens are also expressed by healthy body cells. The dose administered therefore requires attention to keep treatment within safe limits.
• Their short lifespan under conditions in the body limits treatment with CAR NK cells. Findings from recent studies suggest that memory NK cells may be more suitable for CAR NK adoptive cell therapy because of their long lifespan and sustained activity.