February 3, 2023

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Multiple gene editing to construct a new generation of CAR-NK

Multiple gene editing to construct a new generation of CAR-NK


Multiple gene editing to construct a new generation of CAR-NK.


In recent years, CAR engineering of natural killer (NK) cells has achieved considerable development due to the unique biological characteristics of NK cells, making it an extremely attractive field in tumor immunotherapy.

NK cells, even if CAR molecules are expressed through genetic engineering, still retain their inherent ability to recognize tumor cells through their own receptors.

In addition, NK cells do not rely on T cell receptors (TCR) for cytotoxic killing. This makes them more favorable and safer than CAR-T cells.


In the past few years, there has been an unprecedented acceleration in clinical research on CAR-NK cell products.

Throughout the experiment, the researchers used NK cells from different sources and various modular CAR designs to target multiple target antigens. In general, there are two main trends: First, the scope of the target disease has changed.

Although most of the early trials focused on hematological malignancies, many recent trials have begun to explore the use of CAR-NK cell immunotherapy in the treatment of entities.  The effect of tumor.

Second, more and more CAR-NK uses multiple gene editing methods to achieve more complex structural designs to enhance the effectiveness and durability of NK cells. A new generation of CAR-NK has temporarily emerged.


More reasonable structure design


In addition to redirecting CAR-NK cells to different target antigens, recent research focuses have increasingly turned to optimized structural design based on modular combinations of different transmembrane and intracellular costimulatory domains to enhance the effectiveness of CAR-NK .


Nowadays, researchers use existing genetic engineering capabilities to fine-tune the CAR structure to induce a more effective anti-tumor response, increase antigen affinity or extend persistence in vivo.

A variety of costimulatory elements have been studied, including those derived from the immunoglobulin superfamily ( CD28, ICOS ), the TNF receptor superfamily ( 4-1BB, CD27, OX40 and CD40 ), and others including CD40L and toll-like receptors ( TLR ) Domain.

Compared with some early CAR-NK structures that are mainly based on the costimulatory domain involved in T cell activation, the value of NK cell-specific signal adaptors is getting higher and higher, and the effects of DAP10, DAP12 and 2B4 are particularly prominent.


In a preclinical study of CD19-directed CAR-NK, the addition of DAP10 ( physiological adaptor for NKG2D ) enhanced the anti-tumor efficacy compared to the constructs using CD3ζ signal alone.

Similarly, other studies have reported that the addition of DAP12 to the prostate stem cell antigen ( PSCA ) targeted CAR structure, and the addition of 2B4 to the mesothelin-targeted CAR-NK cells, all have amplified anti-tumor efficacy.


Armed CAR


The adjustments of the first three generations of CAR structure design all depend on the physiologically occurring immune cell receptor domains.

The fourth-generation structure, called armed CAR, uses a completely different approach.

By adding molecular payloads, it gives CAR-modified immune cells with additional features and functions that are not found in any physiological immune cell receptor.

This method enables the CAR structure to be engineered to solve some inherent biological limitations of cellular immunotherapy.


Multiple gene editing to construct a new generation of CAR-NK


With the help of modern bicistronic and polycistronic vector platforms, the fourth-generation CAR structure can contain multiple ideal genetic modifications.

The most pressing biological limitation at present is the problem of persistence in the body in the absence of support from exogenous cytokines.

By adding structural transgenic IL-15 cytokines, CAR-NK can maintain the continuous presence in the body and enhance the anti-tumor efficacy.

In addition, by adding an inducible caspase 9 ( iCasp9 ) molecular safety switch, all adoptive NK cells in the circulation can be eliminated to prevent adverse toxicity.


Modification of CAR-NK immune checkpoint


Despite the remarkable success of adoptive NK cell therapy, immune cell depletion is still a treatment obstacle.

In order to build the next generation of CAR-NK, NK cell basic biology is also working hard to determine the negative regulator of NK cell immune function.

It has been found that some checkpoint receptors such as PD-1, LAG-3, TIM-3, TIGIT and KLRG1 are upregulated in depleted NK cells.


Multiple gene editing to construct a new generation of CAR-NK


NKG2A is one of the most significant inhibitory NK cell receptors. Its gene deletion is related to the increased cytotoxicity of NK cells against tumors.

Blocking TIGIT has also been shown to prevent NK cell depletion.

In addition, SH2 protein ( CIS ) can be induced by adding cytokines , which is a key cytokine checkpoint upstream of IL-15 signal, which achieves enhanced metabolic fitness and effector functions in CAR-NK cells.

Other studies include the effects of PD-1/PD-L1 and CTLA-4 blockade on NK cells.


Promote the targeting of CAR-NK cells


In order to achieve a better anti-solid tumor effect, the next-generation CAR-NK needs to be engineered to enhance the ability to migrate and penetrate into the tumor.

Some studies are strengthening the genetic modification of NK cell chemokine receptors so that NK homing to the tumor site more effectively.

In renal cell carcinoma, NK cells modified to express CXCR2 show improved transport to the disease site.

Similarly, forced expression of CXCR4 on CAR-NK cells targeted by EGFRvIII can improve the survival rate of animal models of glioblastoma.


Solid tumors are characterized by a high degree of heterogeneity. Each clone has its own unique immunophenotype and antigen expression pattern.

Single antigen-directed cell therapy often faces huge obstacles. Bispecific and multispecific targeting have become A promising direction to solve the challenges brought about by heterogeneity.


Some studies have been exploring the application of dual-target CAR-NK, such as NK-92-derived CAR-NK cells that are specific to wild-type EGFR and EGFRvIII.

When in a xenograft model, dual-targeted CAR-NK cells effectively inhibited tumor growth and improved survival rate, regardless of the EGRF mutation status.

In addition, a multispecific B7-H3 targeting iPSC-derived CAR-NK can recognize a variety of solid tumors.


Improve immunosuppressive tumor microenvironment


In addition to NK innate immune checkpoints, there are also some external factors caused by the tumor microenvironment, creating a very unfavorable environment for infiltrating immune cells.

These factors include the combined effects of nutritional deficiencies, acidity and hypoxia, as well as the effects of immunosuppressive cells and immunosuppressive factors.


The immunosuppressive metabolite adenosine is known to affect NK cell function by blocking the A2A adenosine receptor ( A2AR ) on the surface of NK cells.

A2AR knockout NK cells showed enhanced tumor control in a xenograft mouse model of BRAF mutant melanoma.

In addition, by using CRISPR/Cas9 to target editing of the TGFBR2 gene, primary human NK cells successfully antagonized the immunosuppressive transforming growth factor β ( TGF-β) .


Regulate CAR-NK metabolism


At present, targeting immune metabolic pathways has become an interesting concept to make NK cells more metabolically vigorous and maintain their functional capabilities.

In NK cells, the production of IFN-γ depends on glucose-driven oxidative phosphorylation.

Once activated, NK cells undergo substantial metabolic changes, thereby increasing the rate of glycolysis and oxidative phosphorylation (OXPHOS) to support the energy required to produce an effective immune response.


Rapamycin complex 1 ( mTORC1 ) plays a key role in regulating the metabolism of NK cells.

Studies have shown that the destruction of CISH signal produces metabolic reorganization of CAR-NK cells, thereby maintaining elevated mTORC1 and MYC activities and promoting the body Persistence and enhanced anti-tumor activity.


At the transcriptional level, sterol regulatory element binding protein ( SREBP ) and MYC have been identified as two central regulatory elements that coordinate metabolic adaptation, enabling activated NK cells to effectively initiate an immune response.

MYC signal up-regulates the expression of glucose transporter and glycolytic enzymes and increases mitosis, adapting the metabolic mechanism to the specific needs of NK cells.

One mechanism for maintaining the level of MYC protein in NK cells is through glycogen synthase kinase 3 ( GSK3 )-mediated phosphorylation and subsequent ubiquitination and proteasome degradation.

Studies have found that inhibition of GSK3 can stabilize NK cells In the MYC level, thereby improving its anti-tumor efficacy.




Many CAR-NK cell therapies using highly innovative next-generation genetic engineering methods are being explored to cope with the unique challenges of solid tumors, and they are expected to enter clinical trials in the short term.

Supporting these developments is a growing understanding of basic immune cell biology, and powerful multifunctional omics and single-cell analysis technologies are increasingly driving our understanding in this area.

With the powerful advancement of genetic engineering capabilities, these insights will drive further treatment innovations, and over time, fundamentally change the current treatment model, significantly improve clinical outcomes, and benefit more cancer patients.



Reference paper:

Multiple gene editing to construct a new generation of CAR-NK

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