June 23, 2021

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What are Emerging methods of CAR-T cell regulation?

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What are Emerging methods of CAR-T cell regulation?

What are Emerging methods of CAR-T cell regulation?



What are Emerging methods of CAR-T cell regulation? As a living medicine, CAR T cells have the potential to activate and proliferate quickly and in large quantities, which contributes to their therapeutic effects, but at the same time it also becomes the basis for the side effects associated with CAR T cell therapy.

The most famous toxicity is called cytokine release syndrome (CRS), which is a systemic inflammatory response characterized by fever, hypotension, and hypoxia (5-7). CRS is triggered by the activation of CAR T cells and the subsequent production of pro-inflammatory cytokines (including IFNγ, IL-6, and IL-2) (8).

This is thought to lead to additional activation of immune and non-immune cells, leading to further production of cytokines, including IL-10, IL-6, and IL-1 (9). The severity of CRS is related to tumor burden, ranging from mild fever to life-threatening organ failure (10, 11).

Neurotoxicity is another serious adverse event that can occur with CRS (12). Although the pathogenesis is unknown, it is believed to be the result of brain endothelial dysfunction (13).

Finally, because very few antigens are truly tumor-specific antigens, if CAR T cells target healthy cells that express and recognize antigens, that is, on the target, they will be toxic outside the tumor. Unfortunately, this has led to serious and fatal results, especially when targeting antigens in solid tumors, hindering the use of CAR T cells in these patients (14-17).

The current clinically approved CAR design cannot control CAR T cells after infusion, so the management of toxicity depends on immunosuppression using systemic corticosteroids and the IL-6 receptor antibody tocilizumab. Unfortunately, the use of immunosuppressive drugs severely limits the durability of CART cells (11).

Considering the severity of toxicity and manufacturing cost, once used in patients, the number and activity of CAR T cells need to be adjusted. In this short review, we describe existing and emerging methods for regulating and controlling CAR T cells, and discuss the advantages and disadvantages of each method.



Passive control

Passive control methods provide a direct opportunity to limit CAR T cell-mediated cytotoxicity, but do not provide downstream control of transplanted cells after blood transfusion (Figure 1, left panel).

What are Emerging methods of CAR-T cell regulation?

Figure 1. Schematic diagram of the three main methods used to control CAR T cells today.

Left: Passive control methods include affinity-adjusted CAR and transient transfection of T cells.

Middle panel: Induction control includes the use of antibodies or suicide induction systems to eliminate CAR T cells. In addition, different drugs have been used to control CAR expression or assemble and divide CAR at the transcriptional level, in which the extracellular and intracellular domains have been separated. Another method is to decouple the binding domain from the intracellular signaling domain, so that the binding adaptor can be provided and titrated.

Right: Autonomous CAR T cells are self-regulating, and can decide whether to initiate or inhibit the cytotoxic killing of target cells based on the surface proteins expressed by healthy cells and cancer cells. CAR, chimeric antigen receptor; TRE, tetracycline response element; TF, transcription factor; SynNotch, synthetic Notch receptor.


Transient transfection

A simple but effective way to regulate CAR T cells is to transiently transfect T cells with mRNA encoding CAR (18-23). Due to the lack of genomic integration, CAR expression is limited by degradation of the mRNA encoding CAR and dilution after each T cell division (18). The result is a steady decrease in the number of CAR-expressing T cells unless new cells are injected. However, due to CAR T cells, repeated infusions are associated with a higher risk of allergic reactions (24). Although the inherent limited persistence of these CAR T cells may affect sustained anti-leukemia effects (25), it also limits long-term hematological toxicity and off-target effects.

Affinity adjustment

Reducing the affinity of the binding domain to the targeted antigen is intended to prevent targeted, non-tumor toxicity in the first place (26, 27). Although affinity-regulated CAR retains the ability to bind to cancer cells with high antigen expression, healthy tissues with lower expression survived (28). Therefore, the use of low-affinity CARs is particularly interesting when targeting antigens that are known to be expressed in small amounts on healthy tissues, such as HER2 or EGFR (26, 27). However, this may also lead to escape variants of cancer cells with low antigen expression (29). In addition, the use of T cell promoters and the level of transduction may lead to heterogeneous expression of CAR protein, so it is difficult to ensure consistent behavior among CAR T cells because their affinity for antigens may be different. A promising strategy to overcome heterogeneous CAR expression is to use the CRISPR/Cas9 system to integrate CAR constructs into the endogenous TCR alpha chain (TRAC) locus (30).




Induced control

Recognizing that CAR T cell toxicity will appear quickly, researchers have developed several exogenous methods to quickly regulate the activity of CAR T cells or completely eliminate them. These methods rely on the co-administration of drugs, so their use depends on the pharmacokinetics, tissue availability, and potential side effects of the selected drug (Figure 1).


Suicide gene

The depletion of CAR T cells can be achieved by designing CAR constructs that also express suicide genes, such as inducible Caspase 9 (iCasp9) (31–39), herpes simplex virus tyrosine kinase (HSV-TK) (40, 41) Or human thymidylate kinase (TMPK) (42). In cells expressing iCasp9 and TMPK, when small molecules are administered, they are eliminated by activating the caspase 3 apoptotic pathway.

The iCasp9 system has been successfully validated in patients undergoing haploidentical stem cell transplantation (HSCT), in which T cells expressing iCasp9 are rapidly removed during the onset of graft-versus-host disease (GvHD) (31). Similarly, administration of ganciclovir to T cells co-expressing HSV-TK can lead to the formation of toxic metabolites, but cell death can take up to several days, as it depends on cell proliferation (40, 41). The use of HSV-TK is severely limited by the high immunogenicity of virus-derived proteins (43).

In addition, as ganciclovir is used as a first-line treatment against cytomegalovirus (CMV) infection, the HSV-TK suicide system becomes complicated, and this virus is often reactivated in HSCT and other immunocompromised patients (44) . Since TMPK and iCasp9 are of human origin, the risk of immunogenicity is low. In fact, long-term implantation for several years in patients injected with iCasp9 expressing cells has been reported (45).


Co-expression of cell surface elimination markers not normally present on T cells allows antibody-mediated degradation and control of CAR T cells (22, 46–52). By using clinically approved antibodies, such as CD20-targeting rituximab (48-50) or EGFR-targeting cetuximab (51, 52), complement or antibody dependence on CAR T cells can be achieved Cytotoxicity (CDC/ADCC) (51). Choosing a marker that is co-expressed on cancer cells allows this method to produce additional tumor killing, but it should be noted that there may be further collateral toxicity.

The cell surface markers also allow positive selection of transduced T cells during the manufacturing process and subsequent monitoring of CAR T cell levels in vivo. However, due to the limited CDC/ADCC capacity of patients receiving chemotherapy before CAR T infusion, the effectiveness of this strategy may be affected (53). In addition, the biodistribution and tissue penetration of antibodies are limited, especially in poorly vascularized tumors (54).

In order to solve these problems, the researchers created an anti-idiotypic CAR (55) that recognizes a mouse CD19-specific CAR (55) or integrated a short peptide epitope called E-tag into the extracellular domain of CAR, and created an anti-E-tag CARs can then be used to eliminate anti-tumor CARs (56).

Since the use of suicide genes and the elimination of markers can lead to the irreversible consumption of this complex treatment, researchers have also developed many reversible methods to control CAR T cells.

Systemic T cell suppression

Current methods of controlling CAR T cells include systemic immunosuppressants, such as corticosteroids (57). The lymphotoxic anti-CD52 antibody alemtuzumab has also been proposed as a method to eliminate CD4 and CD123-specific CART cells (47, 58), because targeting these proteins may lead to blood hypoplasia and toxicity. Recently, studies have shown that the use of tyrosine kinase inhibitor dasatinib can inhibit CAR signaling. Dasatinib inhibits the phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), which is a key component of the T cell signaling pathway.

Preclinical studies have shown that dasatinib treatment can reversibly inhibit CAR T cell proliferation and cytokine production without negatively affecting survival (59, 60). Although dasatinib cannot fully inhibit activated CAR T cells, limiting its use for acute CRS or neurotoxicity, preclinical studies have shown that the drug is superior to dexamethasone in inhibiting further activation (59). Finally, although corticosteroids and alemtuzumab can cause extensive suppression or complete elimination of CAR T cells and healthy lymphocytes, the advantage of dasatinib is that it can act as a faster on/off switch because of its very long half-life. Short, only 4 hours (61).

Adapter-mediated CAR

In order to specifically control the activity of CAR T cells against antigens, several adaptor-mediated CARs have been developed, also known as universal CARs (62-71). A common feature is their tumor recognition method, which is achieved by connecting an adaptor (a molecule recognized by CAR) to an antibody or ligand that recognizes tumor antigens.

Although currently clinically approved CARs are designed to be constitutively active, adapter-dependent CAR T cells can only be identified and killed when the adapter is used, so that CAR T cells can be titratable and reversibly controlled. A major advantage of this method is the ability to target different antigens without the need to redesign and reinfuse T cells.

The adaptor was also designed to use a CD19 fusion protein to redirect the anti-CD19-specific CAR to another target, suggesting that the adaptor protein may be used in CAR T cells that are currently clinically approved (72). However, it has been reported that there are huge differences in adaptor kinetics and subsequent effects on CAR T cells, which may reflect differences in the models used, differences in affinity between adaptors and target cells and CART cells, and the biodistribution of adaptor molecules.

Separate CAR

In addition to directly regulating the binding of CAR to antigen, pharmacological inducers can also be used to control the activity of CAR T cells themselves by separating the extracellular antigen binding domain of CAR from its intracellular signal domain (73, 74).

Therefore, the assembly of a fully functional CAR depends on the administration of dimeric drugs, which limits the activity of CAR through the half-life of the drug. Therefore, CAR T cell activation requires two inputs: tumor antigens and dimerization drugs. Therefore, a split CAR can also be regarded as an AND CAR (see below).

Split-CAR allows temporary and reversible control of the number of functional CARs, but this design cannot prevent toxicity on the target or outside the tumor, because spatial control cannot be achieved due to the lack of control over drug distribution.

Protease inhibitor

It has also been suggested that NS3 protease derived from cleavage of hepatitis C should be strategically incorporated into CAR constructs as a way to control CAR T cell activity. Julelat et al. The NS3 protease is incorporated between the CAR and the degraded part to label the CAR construct for degradation when the NS3 protease inhibitor is administered (75). This construct exhibits reversible and adjustable control of CAR T cell cytotoxicity in vitro. Since NS3 protease is virus-derived, it has immunogenic potential and may limit the persistence of CAR T cells.

TET-On monitoring

Several groups have shown that a drug-induced CAR system can be produced by using the TET-on system to control CAR transcription, thereby achieving reversible control of CAR T cells (76-78). Therefore, CAR mRNA is only produced in the presence of doxycycline, although some background CAR expression has been observed (76). Since the TET-on system is derived from bacteria and viruses, it has significant immunogenic potential, but there is a risk of host-mediated elimination of CART cells. Another disadvantage is the lack of rapid control, if life-threatening side effects occur, because the control occurs at the transcriptional level. However, for highly proliferating CAR T cells that continuously dilute the CAR protein in the daughter cells, transcriptional regulation may be sufficient to limit the number of functional CAR complexes.



Logic gates and autonomous control

As CAR T cell therapy is applied to solid tumors, it is becoming more and more important to distinguish between healthy and malignant tissues (79). Imagine using so-called Boolean logic gates and tumor selective mechanisms to generate autonomous CARs with higher target specificity, which can better distinguish tumor cells from healthy cells (Figure 1, right panel).

AND gate

One way to achieve better decision-making in CAR is to incorporate logical AND gates in order to activate the combination of antigens required. Usually, this dual CAR design consists of two extracellular domains, which are specific to different antigens. Each domain is coupled to different components of the intracellular stimulation device, such as CD3ζ and CD28 or 4-1BB (80- 83).

Such methods have been tested in preclinical prostate and breast cancer models and may allow targeting proteins that are also present in healthy tissues (81, 82). However, it is worrying that even a partial signal through a receptor may generate sufficient T cell activity to cause off-target damage (81, 83).

Therefore, another method is to use only one receptor as the activation signal, and does not have the ability to activate the signal itself. This is achieved using so-called SynNotch receptors, which are coupled to orthogonal transcription factors that are released after binding.

This “priming” results in the expression of a fully functional CAR directed against another cancer-associated antigen, ensuring the local activity of CAR T cells (84, 85). Importantly, in the mouse model, SynNotch CAR does not migrate, but retains its function only in double-positive tumors, indicating that this method ensures good spatial control of CAR (84, 85).

Not gate

It is also possible to better distinguish malignant cells from healthy cells by designing inhibitory CAR (iCAR). iCAR contains a binding domain that specifically binds to antigens expressed on healthy cells. The binding domain is fused to the signal domain of CTLA-4 or PD-1. Therefore, the recognition of healthy antigens leads to an inhibitory signal cascade through dephosphorylation.

The receptor complex covers the activation signal (86). iCAR should limit the activity of CAR T cells to tumor tissues lacking healthy antigens, thereby limiting the activity in target and non-tumor tissues. Such a system may allow the reintroduction of CAR T cells that have previously been shown to cause lethal non-tumor activity, such as ERBB2-specific designs.

Although AND gate and iCAR restrict CAR T cell activity spatially, they cannot control the intensity of CAR T cell activity, nor can they control them in a temporal manner.

Tumor localization mechanism: hypoxia sensitivity and masking CARs

In order to obtain better time control and limit CAR T cell activity to the tumor microenvironment (TME), CAR expression can be controlled by adding hypoxia-inducible factor (HIF) to the CAR construct (87). When CAR T cells are present in a normoxic environment (ie, most healthy tissues), the CAR-HIF construct will continue to degrade, ensuring that CAR expression only occurs under hypoxic conditions, as seen in the TME section.

Since CAR degradation is regulated at the protein level, control is considered to occur quickly, which helps to avoid CAR activity outside the tumor. This method has only been tested in vitro and may not be able to eradicate tumor cells in normoxic tissues, such as the peripheral part of the tumor. In addition, these CAR T cells may show off-target effects in healthy hypoxic tissues (such as bone marrow).

Another way to limit CAR activities to TME is to design a masked CAR, as proposed by Han et al. (88). Here, the CARs antigen binding site is hidden by a masking peptide with a linker sensitive to proteolytic cleavage. The tumor-associated proteases present in TME can then cleave the linker, remove the masking peptide and allow CAR T cells to target antigen-presenting cells.

However, the possibility of endogenous proteases cleaving the masking peptide still exists, paving the way for targeted, non-tumor toxicity.


CAR T cells that produce IL-1 receptor antagonist (IL-1Ra)

One of the central cytokines involved in CRS is IL-1. Javridis et al. Recently, a CAR T cell that constitutively produces IL-1 receptor antagonist was designed to protect mice from CRS-related mortality without affecting anti-tumor efficacy (9). A major advantage of using IL-1 receptor antagonists is that it can cross the blood-brain barrier, which may reduce the neurotoxicity associated with CAR T cells (89).

Although the aforementioned logic gates and hypoxia-sensitive CAR try to enhance tumor specificity, they do not allow clinicians to control CAR T cells, so if life-threatening toxicity occurs, they cannot provide a solution.


Since CAR-related toxicities usually appear quickly, ideally, the control mechanism should allow clinicians to quickly control the activity of CART cells. Due to different research designs, direct comparison of the on/off kinetics of each method becomes difficult, but the regulation of protein levels and the use of suicide genes or elimination markers are expected to be faster than the regulation of transcription levels. Table 1). However, the permanent elimination of CAR T cells will abolish the long-term anti-leukemia effect, so many methods are aimed at reversible control, allowing clinicians to shut down CAR T cells in the event of toxicity.

In the future, a properly designed recombinase-mediated switch will enable CAR activity to be stably switched to the off state by a small molecule, and then flip back to the on state using a second molecule (94). This will enable clinicians to stop CART activity without permanently destroying expensive and life-saving therapeutic products, while avoiding the need to constantly administer inhibitor molecules to maintain the shutdown state.

Ideally, small molecules that have received regulatory approval and show minimal side effects can be selected to quickly and reversibly regulate CAR T cell activity. However, the choice of drugs must also be guided by the type of tumor targeted, because the endothelial barrier, such as the blood-brain barrier, and poor vascularization can prevent proper biodistribution and concentration in the affected organs.


What are Emerging methods of CAR-T cell regulation?


Another method is to completely avoid unwanted immune reactions. This is the basic principle behind many developed logic-gated CAR designs, including iCAR and tumor localization CAR (84, 87). Passive and continuous fine-tuning of CAR expression levels can also be achieved through targeted genome integration (30) or through the use of degrons (95) or synthetic miRNA regulation (96). Not only is this possible to reduce dangerous cytokine release, but reducing CAR expression has also been shown to combat T cell depletion (30).

Cheng et al. recently reported the adaptability of CAR’s recognition and response to soluble ligands (such as secreted cytokines). And create exciting new possibilities in the CAR project (97). CARs targeting immunosuppressive soluble ligands such as TGF-β may help overcome hostile TME, which has proven to be a major obstacle, especially in solid tumors. Similarly, the possible combination of iCAR or SynNotch receptors with the ability to sense inflammatory cytokines can be used to achieve autonomous dynamic feedback control of CAR activity (98).

Therefore, this may lead to the production of CAR T cells, which can respond to increased levels of inflammatory cytokines, thereby preventing the accompanying toxicity. Simultaneous use of inducible and autonomous CAR T cell control methods can significantly improve the safety of CAR treatment.

This can be particularly useful when targeting solid tumors, where the targeted antigen can often be found in other healthy tissues. Although the autonomous CAR control design can limit the cytotoxic activity to the time, location, and target cells of interest, it also includes an inducible kill switch, which will provide additional failure in the case of transplanted T cells that exhibit accidental or oncogenic transformation protection.

Early clinical successes and challenges have led to the explosive growth of new technologies for inducing, autonomous and passive control of CAR T cell functions, providing the community with more and more safe and effective anti-cancer treatment solutions. Ultimately, the required CAR T cell regulation will depend on the location, aggressiveness, and targeting of the tumor.







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