The Fundamentals and Innovation Behind CAR-NK Therapy

A detailed review of NK cell biology and its therapeutic applications through CAR-NK therapy.

Source: Britannica

Abstract

NK cells contain two main types of receptors: KIRs and CLRs. These receptors both have activating and inhibitory variants that activate and inhibit NK cell cytotoxicity respectively. If the signals by the receptors lead favour an activating signal, changes in the cytoskeleton and polarization of the MTOC along with the lytic granules lead to the release of cytotoxic chemicals. This process can be interrupted by the TME where tumors can have higher expression of inhibitory ligands, release immunosuppressive molecules, and recruit inhibitory cells to stop NK cell cytotoxicity. To fix this problem, scientists have come up with CARs, an artificial receptor that allows NK cells to recognize, and kill cancer cells.

Table of Contents:

Introduction

Part I: NK cell Receptors

• 1.1 KIRs
• 1.2 CLRs

Part II: NK Cell Cytotoxicity

• 2.1 Formation of the Immunological Synapse
• 2.2 Movement of Lytic Granules

Part III: Failure of NK Cell Surveillance

Part IV: CAR-NK Therapy

• 4.1 The Functions of CARs
• 4.2 Parts of a CAR
• 4.3 Plasmid Design

Part V: Sources of NK Cells and Clinical Trials

Discussion and Sources

Introduction

Recently, there has been a great push towards trying out newer, more innovative forms of cancer treatment. Immunotherapy is an example of this. This is a form of treatment where scientists are able to alter the immune system to recognize threats such as cancer with better accuracy. As it is activated, the immune system will recognize the cancer and start killing it with our own complex pathway.

CAR-NK therapy is one form of immunotherapy leading the charge toward better outcomes for patients. This method of treatment includes synthesizing a genetic sequence for a CAR (chimeric antigen receptor) which is able to detect antigens that it otherwise wouldn’t be able to. The CARs are then added to natural killer (NK) cells, a member of the immune cells. These cells have been known for their power, as they can be seen as a bridge between our adaptive and innate immune systems.

Before going into the process behind how a CAR is constructed, it’s important to ask: how does an NK cell even work? And how is this process interrupted so that it gives us the reason to start CAR-NK therapy? These questions are answered in this review paper about NK cells and the therapy originating from them.

Part I: NK Cell Receptors

The cell on which the CAR is situated, a natural killer cell, is part of the immune system. This means that its function is to regulate the body and detect any abnormalities. It does this by using sets of receptors going through the cell’s membrane.

There are two types of receptors: activating receptors and inhibitory receptors. The complexities of NK cell activation have been explored for many years and scientists have now come to a well-defined consensus on how the different types of receptors work together to defend the body while also protecting its own cells.

There are two main classes of NK cell receptors: killer cell immunoglobulin receptors (KIRs) and C-type lectin receptors (CLRs).

1.1 KIRs:

Killer cell immunoglobulin receptors primarily recognize the MHC Class-I molecules on the surface of cells, also known as the Human Leukocyte Antigen (HLA) class of molecules. They consist of two to three immunoglobulin-like domains and contain both activating and inhibitory receptors [1]. Interestingly, the extracellular domains on both types of receptors are identical, meaning that they will interact with the same molecule.

It may be thought that the recognition of healthy MHC-1 molecules will now be randomly assigned to activate or inhibit NK response, but this is not the case. It has been determined that inhibitory receptors have more affinity for MHC-1 molecules than activating receptors, and will therefore inhibit the activation of an NK cell [1, 16].

Where these receptors do differ though is in their intercellular domains.

KIRs: Activating Receptors

Although there are still some uncertainties about activating receptor signaling, most fall under the category of ITAMs. This is a shared pathway between NK, T, and B cells which has been studied extensively [3].

Depiction of KIR2DS, an activating KIR (Source: Alphafold)

ITAMs stand for immunoreceptor tyrosine-based activating motifs. In KIRs, this motif is not directly connected to the receptor, but rather associated with it. The activating KIR has either lysine or arginine in its transmembrane domain. These are positively charged amino acids that will associate with the negatively charged aspartate in the transmembrane domain of the intracellular DAP12 adaptor molecule [4]. DAP12 is the molecule that contains the ITAM for signaling.

When there is an interaction between the KIR and a corresponding MHC-I molecule, the KIR and DAP12 will form a receptor-adaptor complex [4]. Upon engagement, the tyrosines in the ITAMs will be phosphorylated by the Src family kinases and will lead to the recruitment of Syk and ZAP-70 [3,4]. This leads to the activation of a complex signaling pathway in which lytic granules, along with cytokines are released from the NK cell.

KIRs: Inhibitory Receptors

The intercellular portion of an inhibitory KIR is what gives its properties. For one, the cytoplasmic tail of the inhibitory receptor is much longer than the short tail of the activating receptor [1]. This is because unlike associating with adaptor proteins containing the signaling motifs, the inhibitory KIRs themselves contain this motif.

Inhibitory receptors contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) which allows it to inhibit an NK cell from releasing its cytotoxins. When the inhibitory receptor engages with its respective ligand, the ITIM will be phosphorylated [1]. However, unlike activating receptors, we do not know the specific kinases that are responsible for this [2].

After phosphorylation, the ITIM can recruit three tyrosine phosphatases: SHIP-1, SHP-1, or SHP-2 [4]. Phosphatases are enzymes that are responsible for the removal of phosphate groups from proteins and molecules in a process called dephosphorylation. After the tyrosine phosphatases have been recruited, they will remove the phosphate groups off of signaling proteins which lead to activation of the NK cell. This allows for the interception of activating signals and the NK cell will not kill the healthy cell.

A summary of KIRs [1]

1.2 C-Type Lectin Receptors

C-type lectin receptors (CLRs) are a second type of receptor expressed on NK cells. They contain the C-type lectin extracellular domain [1]. Like KIRs, they consist of both inhibitory and activating receptors. On NK cells, the CD94/NKG2 family of receptors makes up the majority of CLRs. These are a group of heterodimeric receptors that consist of very well-known signaling pathways.

Instead of interacting with the classical HLA groups such as HLA-A, HLA-B, and HLA-C, these receptors will interact with another subset of HLA molecules, HLA-E.

CLRs: Activating Receptors

In the CD94/NKG2D family, there are two receptors that are considered activators: NKG2C and NKG2E. These receptors are relatively similar in their signaling to activating KIRs as they both recruit the adaptor protein DAP12 to start the ITAM-mediated signaling pathway [2].

CLRs: Inhibitory Receptors

Like the activating receptors, there are two inhibitory CD94/NKG2 family receptors: NKG2A and NKG2B. Again, we see similar patterns between inhibitory motifs between these receptors and inhibitory KIRs.

NKG2D

This receptor seems like it would be part of the CD94/NKG2 family, but it is rather an activating homodimeric CLR [1]. This receptor is special as rather than detecting classical MHC-1 molecules, it is able to detect stress-induced ligands such as MICA [5].

Depiction of NKG2D (Source: Protein Data Bank Europe)

Once recognition occurs, a signaling cascade occurs. Unlike the activating members of the CD94/NKG2 family, the intercellular events are distinct from other receptors. The structure of the transmembrane domain is similar, with a negatively charged aspartate amino acid that associates with an adaptor protein [4]. Unlike KIRs, this adaptor protein is not DAP12, but rather DAP10 [2,3,4,5].

These proteins are distinct from each other as DAP10 does not contain classical ITAMs. It instead contains a similar tyrosine-based signaling motif: YINM [2,5]. The following process is similar to an ITAM, where the motif will be phosphorylated by Src family kinases. After this, there are many pathways the signal can follow. One such pathway is the activation of NK cell cytotoxicity through the recruitment of Vav-1 by Grb2. This leads to the phosphorylation of PLC-γ2 and eventually the activation of cytotoxicity [2].

Pathways for NKG2D signaling [5]

Part II: NK Cell Cytotoxicity

If activating signals overpower inhibitory ones, the NK cell is ready to perform its function. This process contains several steps but can be generalized into two distinct parts: the release of lytic granules and cytokines.

What are Lytic Granules?

Lytic granules are the basis on which NK cells kill their target. They are membranous sacs filled with granzymes and perforin. Unlike T-cells where the lytic granules are formed only after recognition, resting NK cells contain them before activation [7].

These granules are hybrid organelles, which have added components which are the cell-killing cytotoxins. Granzymes A and B, along with perforin are transported into the lytic granule by the trans-Golgi network [7].

The expression of NK cell cytotoxicity can be split into two parts: the preparation of the immunological synapse, and the movement of the lytic granules towards their secretion into the target cell. These parts can further deviate into three parts each.

Formation of Immunological Synapse:

1. Creation of the Synapse
2. Localization of Actin Polymerization
3. Adhesion to Target Cells

Movement of the Lytic Granules:

1. Polarization of the MTOC and Lytic Granules
2. Docking and Fusion of Lytic Granules

2.1 Formation of the Immunological Synapse

Creation of the Synapse

This is the junction between the NK cell and the target cell. The purpose of this synapse is to release the lytic granules. Once an NK cell receptor has signaled for the activation of the NK cell’s cytotoxicity, there are a series of conformational changes that occur for this to happen.

At the point of reception, the junction is formed by the peripheral and central supramolecular activation clusters (pSMAC and cSMAC). The pSMAC is the ring creating the synapse while the cSMAC is the focal point where lytic granules will exit towards the target cell [6].

Localized Actin Polymerization

Following the phosphorylation of the activating motifs, one outcome is an influx of Ca2+ ions [2,3,4]. This is important as calcium facilitates the localization of actin filaments to certain points [8]. Certain proteins, such as the Wiskott-Aldrich Syndrome protein (WASp), also play a role in the polymerization of actin at the lytic synapse. After localizing, actin will form a ring around the pSMAC, leaving the cSMAC open for the polarization of granules to the synapse [6].

Adhesion to Target Cell

This process is characterized by the clustering of high-affinity receptors around the immunological synapse. These proteins, referred to as adhesion proteins, form strong bonds with their corresponding ligands on the target cell [6,7].

An example of these adhesion proteins is LFA-1 and MAC-1 (CD11a and CD11b). Each of these proteins has a respective ligand it binds to; in the case of LFA-1, this ligand is ICAM-1. These receptor-ligand complexes will cluster around the immunological synapse, forming a cell-cell junction that allows for the release of lytic granules into the target cell [7,9].

2.2 Movement of the Lytic Granules

Polarization of Lytic Granules and MTOC

Before lytic granules are brought to the immunological synapse for secretion, they must first be polarized towards the microtubule organizing center (MTOC) [6]. The MTOC is a central hub for the microtubules which allow the lytic granules to travel to the immunological synapse. The most known MTOC is the centrosome, crucial in the process of mitosis. However, the one used in lytic granule polarization is a non-centrosomal MTOC (ncMTOC) [10].

The MTOC does not contain the lytic granules; they must be brought through the microtubule system [6]. After being transported, there are a series of protein interactions that occur that lead to the docking of the lytic granules to the plasma membrane.

The first interaction introduces the Cdc42 interacting protein-4 (CIP4), which is associated with the MTOC. This protein interacts with the microtubules, before interacting with WASp for the polarization of the MTOC towards the immunological synapse. Next, the WASp interacting protein (WIP), associated with lytic granules, polarizes them from the organizing center towards the synapse [6].

Docking and Fusion with Plasma Membrane

The following step is the result of the successful polarization of the lytic granules to the immunological synapse. Proteins that have been identified for their role of docking lytic granules to the plasma membrane are Rab27a, Rab27b, and Myosin IIa [6].

Finally, the fusion of the lytic granules is possible. This process is mediated by SNAREs, special proteins that allow the compartments of the lytic granules to be emptied into the target cell [6].

A summary of NK Cell activation [27]

Part III: Failure of NK Cell Surveillance

One major reason why humans develop cancer and other diseases is the failure of the immune system’s response to the threat. NK cells are no exception to this [12]. The tumor microenvironment plays a major role, in this process [13].

This environment deploys many strategies to prevent the expression of NK cell cytotoxicity. For the purposes of this paper, three reasons will be discussed:

1. Presence of Immunosuppressive Molecules
2. Change in Ligand Expression
3. Overregulation of the Immune System

Presence of Immunosuppressive Molecules

The tumor microenvironment is able to secrete and express various molecules that can subvert the action of NK cells. One such molecule is TGF-β. This molecule is produced by tumor cells and is able to suppress the metabolic activity of NK cells, reducing their cytotoxic effects and tumor-killing capabilities [11].

The pathway of this molecule is well-defined [14]:

1. TGF-β will bind to TGF-β receptor 2 (TGFβR2), leading to a downstream signaling pathway.
2. This receptor will call for the recruitment, transphosphorylation, and activation of TGFβR1.
3. This receptor will lead to the initiation of the SMAD signaling pathway through its phosphorylation by the cytoplasmic kinase domain. It can also lead to a non-SMAD pathway which uses molecules such as RHO and PI3K [28]

Source: [28]

This leads to various downstream events which result in gene influxes. Although the specifics behind how this translates into a slower metabolism are still experimental, there have been several theories, such as the inhibition of cMyc, an important metabolic regulator [14].

Change in Ligand Expression

The ligands that are expressed on a tumor cell play a major role in its detection by NK cells. Normally, these cells will display stress-induced ligands for detection by NKG2D. However, tumor cells can shed these ligands from their cell membrane.

This can be done through metalloproteinases or exosome secretion. This will lead to NKG2D ligands (NKG2DLs) to become soluble, dissociating themselves from the NK cell. Ligands such as MICA, MICB, and ULBP are examples of ligands that can take a soluble form and result in lowered cytotoxicity [11]. Furthermore, tumor cells can also secrete LDH5, which makes healthy cells express these ligands. This introduces chronic activation and the NK cell will reduce the level of NKG2D expressed [11].

Tumor cells can also work in the opposite direction, increasing the levels of inhibitory molecules on their surface. An example of this is the increase of HLA-E found on the surface of tumor cells. This leads to the activation of NKG2A, which inhibits an NK cell’s cytotoxicity.

Overregulation of the Immune System

Despite having inhibitory receptors, immune cells still can be regulated by external cells. While this is helpful to prevent autoimmune diseases, tumor cells are known to recruit these cells to stop an immune response. Three of the biggest suppressors of the immune response are MDSCs, Tregs, and TAMs [15].

Myeloid-derived suppressor cells (MDSCs) are immature cells from the hematopoietic stem cells located in the bone marrow. The TME can recruit these cells and have them produce inhibitory and anti-inflammatory cytokines such as IL-10 to suppress NK cells [15].

T regulator cells (Tregs) are a famous immunosuppressive cell, related to the cytotoxic T cell. They mature in the thymus where they are differentiated from the other variations of T cells. These cells, like MDSCs, also produce IL-10. They produce TGF-β as well, an immunosuppressive molecule discussed earlier [15].

Finally, there are tumor-associated macrophages (TAMs), the most abundant immunosuppressive cells in the TME [17]. They are able to recruit Tregs and MDSCs, along with producing molecules that inhibit the immune response [15].

Part IV: CAR-NK Therapy

CAR-NK therapy takes advantage of the NK cell’s abilities but gives it an artificial boost. It has its roots in CAR-T cell therapy, a treatment that has been used to treat multiple myeloma, acute lymphoblastic leukemia, as well as large B-cell lymphoma.

A chimeric antigen receptor (CAR) is an artificial receptor that is based on the T-cell receptor. It contains several components that can be classified into three main groups: the extracellular, transmembrane, and intercellular domains [18].

4.1 The Function of CARs

The addition of an extra receptor to an NK cell’s already expansive arsenal may seem redundant, but there are logical reasons behind why this works.

The reasons are as follows:

• Specificity: CARs can effectively recognize tumor-associated antigens (TAAs) as they have been genetically engineered from monoclonal antibodies [30]. This allows for better recognition of the antigens, leading to more possibilities for activation [29].
• Stronger Signal: CARs allow for immune cells to have enhanced signaling capabilities, allowing for better activation [30]. In the next section, it will be discussed how the different domains of CARs influence their potency.
• Introduction of Artificial Elements: In recent CARs, they have been designed to release cytokines on activation [18,30]. This allows for better proliferation of effector cells as certain cytokines increase the energy of the immune system.

4.2 Parts of a CAR

Each part of a CAR plays a major role in its success against tumors. As mentioned earlier, CARs are based on existing proteins but are modified to recognize tumor antigens. This activates NK cell cytotoxicity which can eventually eliminate the tumor. The parts of a CAR as is follows:

The Extracellular Domain

This part of the CAR is one of the most important and must be designed very precisely to correctly identify the tumor antigen.

The first part of the extracellular domain is the scFv. This is the part of the CAR based on the immunoglobulin chains located on an antibody. There are two chains: a variable heavy and light chain, as well as a linker. The heavy and light chains bind to the tumor antigens while the linker connects both chains [18].

The hinge domain is the next section and is crucial for the function of the CAR. Without flexibility, the receptor would not be able to “reach” out and bind to tumor antigens. The hinge domain’s function is to provide this flexibility and increase the opportunity for the CAR to bind to the antigen [20].

The Transmembrane Domain

To attach the receptor to the cell, a transmembrane domain is needed to anchor it. This can be done by the use of hydrophobic residues. This is done to match the receptor to the environment of the hydrophobic fatty acid tails. The options for the transmembrane domain include CD3, CD8, CD28, NKG2D, and 2B4 [18].

The Intracellular Domain

This is the last section of the CAR and has gone through many changes through the generations. In the first generation of the CAR, there was just a CD3ζ signaling domain [18]. It has three ITAMs which will cause the downstream events discussed earlier when phosphorylated.

However, first-generation CARs were found to not be very effective at killing tumors. In the second generation CAR, a costimulatory domain was added, with another added in the third. These costimulatory domains act to prevent NK cell exhaustion and keep them functional. Common costimulatory domains include CD28, 4–1BB, and the TNF receptor family of genes [18, 22].

The fourth generation of CARs includes a cytokine secretion gene that allows NK cells to release these messengers at an increased rate. This can lead to better outcomes in CAR therapy but also pose a risk for cytokine release syndrome [18].

The evolution of the activation signals in CAR-NK cells. [18]

4.3 Plasmid Design

After the actual CAR sequence is designed, it is ready to be placed with other elements into a viral vector. This design is crucial as it will be integrated into the cell, meaning it must work cooperatively with the cell’s system.

Viral Vectors: Lentivirus and Retrovirus

The next parts of the plasmid are dependent on the type of vector being used. The decision of which vector to use can depend on many variables, but the main vectors used are lentiviruses and retroviruses.

Lentiviruses include diseases such as HIV-1 and HIV-2. Their affinity for infecting immune cells, such as helper T-cells in HIV, allows for an already established pathway for integration of the CAR within the NK cell [18, 23]. So what separates an actual lentivirus from a lentiviral vector? In short, we are able to take out the bad part of the virus and replace it with what we want.

We can split the virus’ genetic information into two plasmids: one for virus traits we need, and one for the information we want, the transgene. The first plasmid will contain the information for making viral particles and infectivity while the second will have the viral genome without open reading frames (ORFs). These empty ORFs are where the CAR sequence will be added [18].

Retroviruses are a similar case as lentiviruses are a subset of this family of viruses.

Plasmid Backbone and Other Elements

The plasmid backbone is the untranslated region of the viral vector’s sequence. This means that it does not actually code for the protein, but rather has different functions pertaining to the purpose of the protein. In this case, the sequence is coding for a CAR receptor, meaning that there must be high expression, good replication, and a way to track the expression.

The part of the plasmid that asserts high expression of the receptor is the promotor gene. This is the place where RNA polymerase attaches to the plasmid DNA sequence, where it will lead to the start of transcription once the start codon of the CAR sequence is reached [19]. This is followed by the Kozak sequence and the leader peptide. These are sequences that while not part of the functional protein, are important to its translation.

After the CAR sequence itself, there can be different elements added. For example, scientists may want to add a marker to the vector so NK cells expressing the CAR are easy to track. This can be done through various fluorescent proteins such as EGFP. Other elements are also required for the function of the viral vector. For example, the WPRE gene leads to the higher stability of the vector in packaging cells [24].

EGFP is a fluorescent protein that allows researchers to easily see if CARs have been transduced into a cell. Source: Wikipedia

Transduction of Vectors

One advantage that lentiviral vectors have over retroviral ones is that they can pass through the nuclear membrane. This means that it can infect a non-dividing cell, rather than having to wait for the nuclear membrane to disintegrate during mitosis [23].

Lentiviruses are able to pass through the nuclear membrane in a process called nuclear import. While this process is still being studied, the shedding of Caspid protein as the lentivirus enters the cell may allow it to enter through the nuclear pores [23].

Once the virus has entered the nucleus, it is able to associate itself with the DNA of the NK cell and have RNA polymerase read and transcribe the vector. This will eventually lead to the creation of the CAR protein.

Part V: Sources of NK Cells and Clinical Trials

To introduce CAR-NK cells into the patient’s body, there must first be NK cells to transduce with the viral vector. There have been many advancements in this aspect of the therapy, using various sources to get these cells. In this section, some of these sources will be discussed, along with clinical trials that happened or are happening in the world of CAR-NK therapy.

PBMCs

Primary blood mononuclear cells are cells that are found in the patient's own blood. These cells can be extracted, and have the CAR expressed in them. Furthermore, these cells are more mature than their other counterparts, allowing for more cytotoxicity. While this seems good, there are some problems. For one, there would be no standardization of NK cells which would make for a much more difficult situation [25].

NK Cell Lines

A cell line is a cell that can infinitely replicate without stopping. This is especially useful for research purposes when primary cells cannot be used as they provide a cheaper and more efficient method [26].

In CAR-NK therapy, the cell line that is primarily used is the NK92 cell line. Its ability to infinitely replicate makes it a good and fast option for CAR-NK cells. However, these cells are considered cancerous and could harm the patient if they are given like this. Therefore, they must first be irradiated to stop their replication. A side effect of this is lower cytotoxicity and general function [25].

iPSCs

Induced pluripotent stem cells (iPSCs) are a type of cell that can turn into any cell a researcher wants. They are derived from normal somatic cells, where they can undergo certain processes to turn back into a stem cell. In this case, the iPSC could turn into an NK cell. This strategy offers the potential for mass production of CAR-NK cells as they can be manufactured in bulk. However, iPSCs are still very new and also show higher levels of NKG2A than primary NK cells [25].

A pathway for many types of NK cell sources, including more obscure ones not mentioned in this article [25]

Clinical Trials

There have been many clinical trials going on in the world of CAR-NK therapy. Last updated in April 2021, here are a few examples [18]:

• Bari (Front Immunol 2019); Using PBMCs to detect CD19 antigen
• Li (Cell Stem Cell 2018); Using NK92/iPSCs to detect mesothelin
• Chen (Leukemia 2017); Using NK92 to detect CD5

These are simply three of many studies, and many researchers working on CAR-NK therapy to create a better future.

Discussion

While the prospect of treating cancer has long been seen as almost impossible, this therapy gives hope. Its cousin, the CAR-T cell has already transformed the lives of many people, and CAR-NK hopes to achieve the same level of success, even more as well.

Even though the work that has been done is very promising, there are still improvements and things that need to be explored. For example, we’ve already had four generations of CARs; innovation needs to keep happening so obstacles can be overcome. As shown in Part III, the TME is a very complex system that still needs understanding. In other words, we don’t know what we don’t know. To build a CAR that can fight against the TME, it’s necessary to understand it to a detailed degree.

However, this research also has potential downsides to it as well. With newer genetic tools that scientists have at their disposal, it is easier to make irresponsible decisions that can negatively impact humanity. We should continue to progress our current medical technology with innovative solutions like CAR-NK therapy.

Sources

1. Sherif S. Farag, Todd A. Fehniger, Loredana Ruggeri, Andrea Velardi, Michael A. Caligiuri; Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 2002; 100 (6): 1935–1947. doi: https://doi.org/10.1182/blood-2002-02-0350
2. Watzl, C., & Long, E. O. (2010). Signal transduction during activation and inhibition of natural killer cells. Current protocols in immunology, Chapter 11, 10.1002/0471142735.im1109bs90. https://doi.org/10.1002/0471142735.im1109bs90
3. Kumar S. (2018). Natural killer cell cytotoxicity and its regulation by inhibitory receptors. Immunology, 154(3), 383–393. https://doi.org/10.1111/imm.12921
4. Lanier L. L. (2008). Up on the tightrope: natural killer cell activation and inhibition. Nature immunology, 9(5), 495–502. https://doi.org/10.1038/ni1581
5. Siemaszko, J., Marzec-Przyszlak, A., & Bogunia-Kubik, K. (2021). NKG2D Natural Killer Cell Receptor-A Short Description and Potential Clinical Applications. Cells, 10(6), 1420. https://doi.org/10.3390/cells10061420
6. Topham, N. J., & Hewitt, E. W. (2009). Natural killer cell cytotoxicity: how do they pull the trigger?. Immunology, 128(1), 7–15. https://doi.org/10.1111/j.1365-2567.2009.03123.x
7. Orange J. S. (2008). Formation and function of the lytic NK-cell immunological synapse. Nature reviews. Immunology, 8(9), 713–725. https://doi.org/10.1038/nri2381
8. Hartzell, C. A., Jankowska, K. I., Burkhardt, J. K., & Lewis, R. S. (2016). Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse. eLife, 5, e14850. https://doi.org/10.7554/eLife.14850
9. Shannon, M. J., & Mace, E. M. (2021). Natural Killer Cell Integrins and Their Functions in Tissue Residency. Frontiers in immunology, 12, 647358. https://doi.org/10.3389/fimmu.2021.647358
10. Sanchez, A. D., & Feldman, J. L. (2017). Microtubule-organizing centers: from the centrosome to non-centrosomal sites. Current opinion in cell biology, 44, 93–101. https://doi.org/10.1016/j.ceb.2016.09.003
11. Lian, G., Mak, T. S., Yu, X., & Lan, H. Y. (2021). Challenges and Recent Advances in NK Cell-Targeted Immunotherapies in Solid Tumors. International journal of molecular sciences, 23(1), 164. https://doi.org/10.3390/ijms23010164
12. Seliger, B., & Koehl, U. (2022). Underlying mechanisms of evasion from NK cells as rationale for improvement of NK cell-based immunotherapies. Frontiers in immunology, 13, 910595. https://doi.org/10.3389/fimmu.2022.910595
13. Anderson, N. M., & Simon, M. C. (2020). The tumor microenvironment. Current biology : CB, 30(16), R921–R925. https://doi.org/10.1016/j.cub.2020.06.081
14. Slattery K and Gardiner CM (2019) NK Cell Metabolism and TGFβ — Implications for Immunotherapy. Front. Immunol. 10:2915. doi: 10.3389/fimmu.2019.02915
15. Tie, Y., Tang, F., Wei, Y. Q., & Wei, X. W. (2022). Immunosuppressive cells in cancer: mechanisms and potential therapeutic targets. Journal of hematology & oncology, 15(1), 61. https://doi.org/10.1186/s13045-022-01282-8
16. Stewart, C. A., Laugier-Anfossi, F., Vély, F., Saulquin, X., Riedmuller, J., Tisserant, A., Gauthier, L., Romagné, F., Ferracci, G., Arosa, F. A., Moretta, A., Sun, P. D., Ugolini, S., & Vivier, E. (2005). Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors. Proceedings of the National Academy of Sciences of the United States of America, 102(37), 13224–13229. https://doi.org/10.1073/pnas.0503594102
17. Cheng, N., Bai, X., Shu, Y., Ahmad, O., & Shen, P. (2021). Targeting tumor-associated macrophages as an antitumor strategy. Biochemical pharmacology, 183, 114354. https://doi.org/10.1016/j.bcp.2020.114354
18. Gong, Y., Klein Wolterink, R. G. J., Wang, J., Bos, G. M. J., & Germeraad, W. T. V. (2021). Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. Journal of hematology & oncology, 14(1), 73. https://doi.org/10.1186/s13045-021-01083-5
19. Juven-Gershon, T., & Kadonaga, J. T. (2010). Regulation of gene expression via the core promoter and the basal transcriptional machinery. Developmental biology, 339(2), 225–229. https://doi.org/10.1016/j.ydbio.2009.08.009
20. Hudecek, M., Sommermeyer, D., Kosasih, P. L., Silva-Benedict, A., Liu, L., Rader, C., Jensen, M. C., & Riddell, S. R. (2015). The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer immunology research, 3(2), 125–135. https://doi.org/10.1158/2326-6066.CIR-14-0127
21. Zhang, J., Hu, Y., Yang, J., Li, W., Zhang, M., Wang, Q., Zhang, L., Wei, G., Tian, Y., Zhao, K., Chen, A., Tan, B., Cui, J., Li, D., Li, Y., Qi, Y., Wang, D., Wu, Y., Li, D., Du, B., … Huang, H. (2022). Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature, 609(7926), 369–374. https://doi.org/10.1038/s41586-022-05140-y
22. Elahi, R., Heidary, A. H., Hadiloo, K., & Esmaeilzadeh, A. (2021). Chimeric Antigen Receptor-Engineered Natural Killer (CAR NK) Cells in Cancer Treatment; Recent Advances and Future Prospects. Stem cell reviews and reports, 17(6), 2081–2106. https://doi.org/10.1007/s12015-021-10246-3
23. Durand, S., & Cimarelli, A. (2011). The inside out of lentiviral vectors. Viruses, 3(2), 132–159. https://doi.org/10.3390/v3020132
24. Zufferey, R., Donello, J. E., Trono, D., & Hope, T. J. (1999). Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. Journal of virology, 73(4), 2886–2892. https://doi.org/10.1128/JVI.73.4.2886-2892.1999
25. Xie, G., Dong, H., Liang, Y., Ham, J. D., Rizwan, R., & Chen, J. (2020). CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine, 59, 102975. https://doi.org/10.1016/j.ebiom.2020.102975
26. Kaur, G., & Dufour, J. M. (2012). Cell lines: Valuable tools or useless artifacts. Spermatogenesis, 2(1), 1–5. https://doi.org/10.4161/spmg.19885
27. Cantoni, C, Wurzer, H, Thomas, C, Vitale, M. Escape of tumor cells from the NK cell cytotoxic activity. J Leukoc Biol. 2020; 108: 1339–1360. https://doi.org/10.1002/JLB.2MR0820-652R
28. Zhang, M., Zhang, Y. Y., Chen, Y., Wang, J., Wang, Q., & Lu, H. (2021). TGF-β Signaling and Resistance to Cancer Therapy. Frontiers in cell and developmental biology, 9, 786728. https://doi.org/10.3389/fcell.2021.786728
29. Marco L. Davila, Renier Brentjens, Xiuyan Wang, Isabelle Rivière & Michel Sadelain (2012) How do CARs work?, OncoImmunology, 1:9, 1577–1583, DOI: 10.4161/onci.22524
30. S. E. Lindner et al., Chimeric antigen receptor signaling: Functional consequences and design implications.Sci. Adv.6,eaaz3223(2020).DOI:10.1126/sciadv.aaz3223