April 18, 2024

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Challenges and future prospects of CAR-NK cell therapy

Challenges and future prospects of CAR-NK cell therapy


Challenges and future prospects of CAR-NK cell therapy.

T cell therapy based on chimeric antigen receptors (CARs) has shown great success for blood cancer;

however, potentially fatal toxicity (such as cytokine release syndrome) and high cost limit the clinical application of CAR-T cells Some shortcomings need to be overcome.


Natural killer (NK) cells are the focus of current immunological research. So far, the use of NK cells to treat malignant tumors has achieved certain success.

The latest advances in NK cell receptor biology have greatly changed our understanding of how NK cells recognize and kill tumor cells.

CAR-NK cells have become a strong candidate for cancer retargeting therapy due to their unique recognition mechanism, strong cytotoxicity and clinical safety.

In addition, NK cells reduce the risk of allogeneic reactions and can be used as “ready-made products.”


Although CAR-NK therapy in full swing still faces many challenges and obstacles, its huge potential has shown broad prospects in the field of tumor immunotherapy.




NK cell biology


NK cells are the first subtype of innate lymphocytes (ILC) to be identified , which can produce a variety of effector functions on virus-infected and/or transformed cells, mainly cell killing and production of pro-inflammatory cytokines.

NK cells and other ILC family members (ILC1s, ILC2s, and ILC3s) are derived from the same lymphoid progenitor cells as B cells and T cells. The cytotoxic activity of NK cells makes them the most functionally similar to CD8+ T cells, and the cytokine production patterns of the ILC1, ILC2 and ILC3 populations correspond to the TH1, TH2 and TH17 subpopulations of CD4+ T cells, respectively.


The two most typical subpopulations of NK cells are the CD56brightCD16- and CD56dimCD16+ populations.

The number of CD56bright cells in peripheral blood is relatively small (90% of circulating NK cells are CD56dim) , while the NK cells in tissues are mainly CD56bright.

CD56bright NK cells are powerful cytokine producers.

Unless stimulated by pro-inflammatory cytokines such as IL-15, their cytotoxicity is weak. In contrast, CD56dim NK cell population can mediate the continuous killing of infected and malignant cells, mainly through exocytosis of pre-assembled cytolytic particles containing granzyme B and perforin in the immune synapse, and finally induce target cells Apoptosis.




Activating and inhibitory receptors of NK cells

Unlike B cells and T cells, NK cells do not express somatically rearranged antigen receptors, but a random combination of activated receptors and inhibitory receptors.

The balance of stimulation signals and inhibitory signals through these different receptors produces a response or tolerance to target cells. MHC-I (major histocompatibility complex class I) antigen-specific inhibitory receptors can closely regulate NK cell-mediated cytotoxicity and lymphokine production.

The inhibitory signal of MHC-I specific receptor is essential for hematopoietic cells to avoid the destruction of NK cells.

This concept is called “lost self” and was originally proposed by Ljunggren and Karre.

This MHC-I recognition inhibitory receptor forms three families of NK cell surface receptors, namely KIRs (killer cell immunoglobulin-like receptors) , LIRs (leukocyte immunoglobulin-like receptors) and NKG2A (natural killer) Cell Group 2 A) .


Challenges and future prospects of CAR-NK cell therapy



KIRs are members of the immunoglobulin superfamily and are type I transmembrane molecules that recognize classic human leukocyte antigens A, B, and C (HLA-Ia) . LIRs, also known as ILTs (immunoglobulin-like transcripts) , form a second set of receptors, in addition to HLA Ia, they mainly recognize non-classical HLA-G (Ib) molecules. LIRs and KIRs belong to the same Ig superfamily.

NKG2A is a member of the NKG2 family, including A, B, C, D, E, F, and H, and dimerizes with CD94 to form the NKG2A/CD94 receptor.

It belongs to the C-type lectin family of receptors and recognizes non-classical HLA-EⅠ molecules as its ligands.


The killing effect of NK cells requires not only the detection of MHC-I molecules on transformed cells through inhibitory receptors, but also the activation of NK cells through activating receptors. Natural cytotoxic receptors (NCR) are a group of activating receptors on the surface of natural killer cells, including NKp46, NKp30 and NKp44.

These receptors as well as NKG2D and DNAM-1 (DNAX helper molecule-1) recognize ligands expressed on the surface of virally infected or malignantly transformed cells. Some co-receptors (2B4, NKp80, NTB-A and CD59) are also expressed, and they can only play a role in combination with other activating receptors.

CD16 (or FcγRIII) is also an activating receptor, which is mainly expressed by CD56dim NK cell subsets, which is essential for the antibody-dependent cytotoxicity (ADCC) of IgG-coated target cells .





CAR-NK cells and CAR-T cells have extracellular, transmembrane and intracellular signal transduction domains.

NK cells increase their cytotoxicity and cytokine production through two other costimulatory molecules, namely NKG2D and CD244 (2B4) .

Therefore, it has stronger tumor-specific targeting and cytotoxicity than CAR-T cells. CAR-NK cell therapy may become an alternative to CAR-T therapy in the future, because CAR-NK cells have the following unique characteristics that surpass CAR-T.


Challenges and future prospects of CAR-NK cell therapy



First, allogeneic NK cells are quite safe for adoptive cell therapy (ACT) because they usually do not mediate GVHD.

In addition, NK cells secrete only a small amount of IFN-γ and GM-CSF, and do not produce IL-1 and IL-6 that initiate CRS.

Secondly, in addition to inhibiting cancer cells by recognizing tumor surface antigens by single-chain antibodies, NK cells can also inhibit cancer cells by recognizing various ligands through a variety of receptors, such as natural cytotoxic receptors (NKp46, NKp44, and NKp30) , NKG2D and DNAM-1 (CD226) .

Finally, NK cells are very abundant in clinical samples and can be produced from peripheral blood (PB) , umbilical cord blood (UCB) , human embryonic stem cells (HESC) , induced pluripotent stem cells (iPSC) and even NK-92 cell lines.




Clinical research on CAR-NK cell therapy


The research on CAR-NK therapy is currently in its infancy, and the number of clinical studies is increasing year by year.

In addition, in terms of research targets, the CD19 antigen is the most common in hematological tumors.

The most widely advanced CAR-NK therapy in solid tumors includes targets such as tumor-associated antigens HER2, MUC1 and PMSA.


Currently, some ongoing clinical trials are investigating the safety and effectiveness of CAR-NK cells in the treatment of hematological tumors and solid tumors. These trials are listed in the table below.


Challenges and future prospects of CAR-NK cell therapy




Challenges of CAR-NK cell therapy


Low durability

In the absence of cytokine support, the lack of in vivo persistence of infused cells is one of the main disadvantages of adoptive NK cell therapy.

Although it may be safer, it will also limit the effectiveness of NK cell immunotherapy.

Exogenous cytokines have been shown to increase the proliferation and durability of adoptive NK cells; however, they may also cause undesirable side effects, including the growth of suppressive immune subpopulations, such as Tregs.


Metastasis to the desired tumor site

Quick homing to the tumor bed is essential for the effect of adoptive cell therapy, and is controlled by the complex interaction between NK cells and chemokines released by tumor cells.

However, the efficiency of NK cells homing to tumor sites has been controversial, which has prompted continuous efforts to improve.


Some researchers have studied various engineering methods to improve NK cell homing.

For example, NK cells are electroporated with mRNA encoding the chemokine receptor CCR7 to increase migration to lymph nodes that express the chemokine CCL19.

NK cells transduced with a viral vector encoding CXCR2 showed better motility for renal cell carcinoma tumors expressing homologous ligands such as CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8.

In order to improve the success rate of NK cell immunotherapy for patients with solid tumors, several new technologies that promote the transport of NK cells to tumor sites have been studied in mouse models; however, the effectiveness of these methods needs to be verified in clinical trials.


Immunosuppressive tumor microenvironment

TME includes immunosuppressive molecules, immunosuppressive cells, and an unfavorable environment that hinders the function of immune cells, and is the main obstacle to CAR-NK cell therapy.

TGF-β; Adenosine; Indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) are immunosuppressive cytokines and metabolites found in TME that can damage the activity of NK cells.

Treg cells; regulatory B cells; myeloid-derived suppressor cells; tumor-associated macrophages (TAM) ; platelets; fibroblasts and some unfavorable metabolic factors such as hypoxia, acidity and nutritional deficiencies, which can cause in a malignant environment Immunosuppressive.


Therefore, researchers are working to develop CAR-NK cells that can prevent certain immunosuppressive effects. For example, by using CRISPR/Cas9 technology to knock out the TGF-βR2 gene or block the high-affinity A2A adenosine receptor on NK cells.

Another important method for TME to deplete NK cells is immune checkpoint molecules. To overcome this problem, genome editing is used to eliminate the checkpoint components of NK cells to improve their functions.


Low transduction efficiency of lentivirus

The lentivirus-based transduction system is one of the most commonly used methods for intracellular gene modification and delivery.

However, due to natural characteristics, NK cells are resistant to lentivirus, which makes lentivirus-based transduction a challenge. In order to improve virus transduction, various chemicals are used.

For example, protamine sulfate or dextran can be used to remove the charge on the cell membrane.




Future prospects of CAR-NK cell therapy


Recognize the target antigen

The most critical step in the design of CARs is to identify highly uniformly expressed target tumor antigens.

Most tumor-associated antigens (TAAs) are also expressed by some healthy cells, so it is inevitable to bring about “targeting non-tumor” effects.

In addition, there may be huge differences in the expression of these TAAs in single-cell clones of the same tumor. In order to overcome this problem, people have designed a bispecific CAR that can target multiple antigens at the same time.


This can be achieved in a variety of ways. For example, different CAR-NK cells targeting different antigens can be injected at the same time; or a CAR that can recognize multiple antigens can be designed.

This goal can be achieved by “tandem CAR”, in which two combine Dots are connected to a single molecule to increase the efficiency of immune synapses.

In addition, multiple CARs can be produced on the same immune cell by using a vector called “bicistronic CAR”.


Improve NK cell activity

Several immune checkpoints regulate and inhibit NK cell activity.

These immune checkpoints act as a “natural brake” to prevent autoimmune diseases or immune pathological conditions caused by excessive activation.

Gene deletion or blockade of these checkpoints can help CAR-NK cells remain overactive and get rid of cancer and metastasis faster.


For example, a new NK-92 cell line is designed to target PD-L1, IL-2 retained by ER, and a high-affinity CD16 CAR, called PD-L1 targeting haNK (t-haNK) .

Exciting preclinical data show that these cells have specific anti-tumor effects on 15 tumor cell lines in vitro, and have strong anti-tumor effects on triple-negative breast cancer, bladder tumors and lung cancer in vivo.


Another important strategy to improve the activity of CAR-NK cells is the regulation of tumor metabolism, but this strategy has not yet received the attention it deserves.

Under hypoxic conditions, adenosine is produced by the metabolism of ATP through CD39 and CD73. They participate in immune evasion, prevent the transport of NK cells to the tumor site, and prevent the maturation of NK cells.

NKG2D engineered CAR-NK cells showed good effects in treating lung cancer after being inhibited by anti-CD73 antibodies.


Overcome the immunosuppressive microenvironment

Tumors have a variety of immunosuppressive factors, such as TGF-β, IL-10, PD-1 or arginase.

There are several ways to reduce the inhibitory effect of TGF-β. For example, the combination of a TGF-β kinase inhibitor and NK cells was found to restore the cytotoxicity of NK cells and preserve the expression of NKG2D and CD16.

In addition, the use of hybrid CARs with extracellular TGF-β receptor domains has been found to be quite successful in improving the anti-tumor potential of NK-92 cells.

By knocking out SMAD3 in solid tumors, the cytotoxic activity of NK cells has been enhanced.


Improve safety

Important methods to improve the safety of CAR-NK cell-based therapy may include modifying the CAR structure by adding suicide genes or developing bispecific CAR molecules to better target tumor-specific antigens.


CAR-NK cells can target tumors in both CAR-dependent and CAR-independent ways; therefore, this feature of NK cells can be used to exert enhanced tumor killing effects and develop non-signaling CARs.

These non-signal CARs lack direct killing signals, but by promoting the resident and adhesion of NK cells on target cells, they can enhance the specific killing of NK cells. Another interesting strategy is to design CAR-NK that can regulate TME.

This CAR-NK is named “armored” CAR-NK cells. These very special CAR-NK cells express several foreign genes, which can regulate local TME to prevent any harmful effects.


Improve accessibility

In order to overcome the accessibility of CAR-NK cells in solid tumors, several methods can be used, including local administration, intraperitoneal administration and focused ultrasound-guided administration.

For example, in an orthotopic model that simulates human pleural malignancies, it is found that pleural injection is very effective, and its function duration is even longer than that obtained by intravenous injection.

Local administration of CAR immune cells may also help reduce the therapeutic dose.






In general, the progress and progress in the field of NK cell immunobiology has laid the foundation for better and more novel immunotherapy.

The excellent anti-tumor effect of NK cells has made it the focus of cellular immunotherapy.

CAR-NK cell therapy is a promising field of clinical research. Compared with CAR-T cells, CAR-NK cells have their own unique advantages, but they still face some challenges.

These challenges include cell persistence, overcoming the immunosuppressive microenvironment, and transduction efficiency. 

We believe that solving these problems, based on the excellent anti-tumor pedigree of NK cells, is very likely to bring new breakthroughs in tumor treatment under the arm of CAR modification.






References :

1.CAR-NK Cells: From Natural Basis to Designfor Kill. Front Immunol. 2021; 12: 707542.

Challenges and future prospects of CAR-NK cell therapy

(source:internet, reference only)

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