April 12, 2024

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Strategies to deal with CAR T cell treatment obstacles

Strategies to deal with CAR T cell treatment obstacles


Strategies to deal with CAR T cell treatment obstacles. CAR T cell therapy has achieved great success. However, its adverse events, such as cytokine release syndrome (CRS), neurotoxicity, graft rejection, target non-tumor toxicity and tumor recurrence, have restricted the rescue of CAR-T cell therapy.

In addition, in the case of solid tumor treatment, CAR T cell therapy has not produced encouraging results, mainly due to the powerful tumor microenvironment (TME) network that acts in an inhibitory manner to cause CAR-T cell dysfunction. Kind of challenge.


In this article, we tend to describe the above considerations in detail, and then discuss smart strategies to eliminate these considerations. In addition, we briefly introduced how this well-designed strategy makes possible safer and more effective CAR T cell therapy.



1. Anti-CRS and neurotoxic drugs

CRS is the most common side effect of CAR T cell therapy and is usually observed a few days after adoptive transfer (28, 33). The usual features of CRS are IL-1, IL-2, IL-6, IL-8, IL-10, interferon-γ (INF-γ), granulocyte-macrophage colony stimulating factor (GM-CSF), And tumor necrosis factor alpha (TNF-α) in the patient’s serum (28, 33).

The starting point of the so-called storm is the activation of CAR T cells after binding to the target antigen (28, 33). This activation causes CAR T cells to produce and secrete inflammatory cytokines (28, 33). In response to these cytokines, other innate immune cells, such as macrophages, begin to release inflammatory cytokines, such as IL-1 and IL-6, thereby forming an inflammatory loop (28, 33). In order to keep the situation under control, the self-reinforcing cycle mentioned needs to be interrupted.


Researchers report the presence of CD19-redirected CAR T cells in the cerebrospinal fluid of patients and elevated levels of pro-inflammatory cytokines (28, 34). In addition, high concentrations of this cytokine may activate the cerebral vascular endothelium and the blood-brain barrier (BBB), leading to their permeability and subsequent brain edema (35). According to the latest report by Parker and colleagues, in the case of CD19-based CAR T cell therapy, the observed neurotoxicity can be attributed to the targeting effect of CAR T cells on CD19-expressing brain wall cells (36).
In this section, we briefly discuss strategies that may benefit CAR T cell-mediated CRS and neurotoxicity treatments.


GM-CSF blockade

GM-CSF is a cytokine that activates macrophages and monocytes, and is known to be an important factor in mediating CRS (28, 29). GM-CSF can be neutralized with mAbs (such as lenzilumab), which can lead to a significant reduction in myeloid and T cell infiltration in the central nervous system (CNS) (37). This reduction helps to alleviate neuroinflammation (NI) in preclinical models and prevent CAR T cell-mediated CRS (37). In addition, this method not only does not interfere with the function of CAR T cells, but can also improve its killing effect by reducing the risk of CAR T cell-mediated CRS and NI (37).
In detail, the neutralization effect of GM-CSF can inhibit the secretion of CRS-causing cytokines (such as IL-6) and reduce other CRS-mediated pro-inflammatory factors (including IL-8 and monocyte chemoattractant protein 1 ( MCP-1)). As an immune cell transport mediator (38, 39).

This method reduces the level of key CRS mediators and enhances the anti-tumor activity of CAR T cells in preclinical models (37). Most importantly, CAR T cells can also be genetically engineered to secrete GM-CSF neutralizing antibodies, thereby further reducing the risk of CRS and neurotoxicity.


IL-1 and IL-6 blockade

Studies have shown that IL-1 and IL-6 released by monocytes and macrophages are related to CAR T cell-mediated CRS and immune effector cell-related neurotoxicity syndrome (ICANS) (41, 42). Preclinical data indicate that during the onset of CRS, monocytes are the main source of IL-1 and IL-6 (42). It has been shown that CRS can be prevented by using tocilizumab for monocyte ablation or IL-6 receptor blockade (42). However, tocilizumab has been reported to fail to prevent delayed lethal neurotoxicity in preclinical mouse models (42). In this case, Anakinra is an immunosuppressive drug and IL-1 receptor antagonist. After administration in a preclinical mouse CRS model, it has been shown to protect them from lethal neurotoxicity and CRS. Gave encouraging results (42).
In short, IL-1 and IL-6 are both key factors in the occurrence and development of CRS and neurotoxicity after CAR T cell infusion (42).


Catecholamine blockade

Recently, it has been found that high levels of circulating catecholamines can mediate various types of immune disorders, including CRS, through the self-reinforcing loop in macrophages (45). Catecholamines have an effective role in the release of cytokines induced by T cell activation therapeutics (45). It has been found that inhibiting the synthesis of catecholamines can lead to a significant reduction in the level of cytokine release in vivo and in vitro (45). Such studies have shown that catecholamines are key regulators of cytokine release, not only blocking their synthesis pathways will not cause side effects or impaired CAR T cell function, but also can reduce the incidence and development of CRS (45, 46).



2. Resistance to immune rejection (this part is for allogeneic CART)

Allogeneic T cells (obtained from healthy donors) may be rejected by the recipient’s immune system (28, 29). This adverse event is mainly mediated by the recipient’s T cells and natural killer (NK) cells, because these cells recognize allogeneic CAR T cells as invading foreign cells and should be eliminated from the host (47-51).


Alloimmune Defense Receptor (ADR)

One of the latest strategies to solve the problem of allogeneic CAR T cell rejection is to use the 4-1BB cell surface receptor present on the receptor T cells and NK cells (51). The expression of this receptor is up-regulated in activated T cells and NK cells (51). This strategy uses an engineered receptor called Alloimmune Defence Receptor (ADR), which is a 4-1BB recognition domain derived from a 4-1BB ligand (4-1BBL), intracellular CD3ζ domain, spacer and transmembrane Structure domain (51). ADR is designed to be expressed on the surface of CAR T cells (51). Specifically, ADR recognizes activated alloreactive T cells and 4-1BB molecules up-regulated on the surface of NK cells, which leads to the activation of CAR T cells expressing ADR and eliminates the receptor’s alloreactive immune cells ( 51). Moreover, the expression of ADR does not affect the effector function of CART cells. Therefore, this method can provide a new weapon for allogeneic CAR T cells, which can be used to resist immune cells in an attempt to interfere with their anti-tumor effects (51) .
Figure 2 shows a detailed description of CAR T cells expressing ADR.

Strategies to deal with CAR T cell treatment obstacles


CD47 expression

CD47 is a transmembrane protein responsible for mediating the “don’t eat me” signal in many types of malignant cells (55). Signal regulatory protein-α (SIRPα) is considered to be the receptor for CD47 on various immune cells including macrophages (55). Once tumor cells expressing CD47 antigen on their surface encounter macrophages, CD47 binds to SIRPα, leading to the spread of the “don’t eat me” signal, and thus eliminate the phagocytosis of macrophages (55). Therefore, using this mechanism, malignant cells can easily evade immune system-mediated eradication (55). This mechanism can be applied when allogeneic CAR T cells are used to avoid macrophage-assisted CAR T cell rejection and subsequent clearance. In this regard, CAR T cells can be modified to express CD47 on their surface to avoid the phagocytosis of macrophages.
Knock out TCR and HLA

The researchers also used genetic engineering methods to reduce the level of allogeneic response when using allogeneic CAR T cells. In this regard, the TRAC gene is one of the most important targets. A variety of genetic manipulation strategies (such as TALEN, Zinc Finger Nuclease (ZFN) and CRISPR-Cas9) have been used to knock out genes, which can effectively eliminate these two genes TCRα and β chain and alleviate homoreactivity (56-60). In addition, other researchers emphasized the use of CART cells of the same species with TCR and CD52 gene knockout functions, and demonstrated that these cells can be satisfactory general CAR T cell candidates because they do not cause allogeneic reactions and can R/RB ALL is used to mediate molecular remission of patients (57). It is worth mentioning that CD52 knockout makes these CAR T cells resistant to the depletion of the anti-CD52 antibody alemtuzumab (57). In addition, other researchers have also studied CRISPR-Cas9-mediated CAR transgene knock-in in the TRAC gene locus, because they believe that this method can also be the same as other methods mentioned to disrupt endogenous TCR expression in allogeneic CART cells. Effective (61). Similarly, both CRISPR-Cas9 and ZFN have been used to ablate HLA expression to reduce the level of allogeneic response when using allogeneic CAR T cells (62, 63).



3. Strategies to overcome tumor toxicity outside the target

Even at lower rates, TAA targeted by CAR T cells is usually expressed in healthy tissues. However, despite the limited expression of target antigens, CAR T cells still manage to recognize these normal cells and initiate a cytolytic response against them. This phenomenon leads to the elimination of healthy cells (known as “off-target non-tumor” toxicity), leading to life-threatening side effects, such as multiple organ failure for individual patients. In order to solve these limitations, scientists have designed a smart CAR construct with a tumor-selective mechanism that can accurately distinguish malignant and healthy cells. In this section, we will briefly discuss some of these strategies, while highlighting their advantages and disadvantages.


“Hidden” CAR

Conditionally active CAR constructs (the antigen recognition domain of which is composed of precursors) constitute a new strategy for “concealment CAR”, thereby increasing the applicability of CAR T cells in the treatment of cancers lacking definitive TAA (73). Specifically, a precursor antibody is an antibody whose antigen recognition site is covered by a masking peptide recombined with a protease-sensitive linker. The protease-sensitive linker is only prone to proteolytic cleavage by TME protease (73, 74) . Conceptually, protease-sensitive linkers are cleaved in the presence of tumor-associated proteases, causing subsequent masking peptides to break away and expose the antigen binding site of the targeting domain (73).
This situation opens the door for the downstream tumor-killing response of effector cells (Figure 3A) (73). Compared with conventional mAbs, due to their extended pharmacokinetic half-life, their safety index has been greatly improved, which allows them to achieve higher exposure rates when reaching the same dose level as conventional mAbs (74). This extended safety zone may be translatable in the field of covert CART cell therapy, in that a higher infusion dose can impart a more effective therapeutic effect without crossing the safety red line (73).

Strategies to deal with CAR T cell treatment obstacles


Inhibitory CAR (iCAR)

Another strategy to minimize the harmful damage of “off-target tumor toxicity” or damage to bystander’s healthy tissue is to use antigen-specific iCAR. The general concept is to fuse the surface antigen recognition domain with the signal domain of an endogenous immunosuppressive receptor (such as CTLA-4 or PD-1), thereby reversibly restricting the secretion, cytotoxicity and proliferation of T cell cytokines, although activation is affected. Participate at the same time (it can be CAR or just engineering TCR) (75). The iCAR platform allows T cells to differentiate between healthy cells and cancer cells in an antigen-selective manner (Figure 3B) (75). In the absence of its specific inhibitory antigen, the transgene expression of the iCAR construct will not affect the basic functions of T cells (75). In addition, other T cell restricted inhibitory receptors, such as BTLA, 2B4 and LAG-3 or their combination in a single second-generation iCAR or as an iCAR with multiple combined cytoplasmic domains, can also be used to modulate CAR T The cytotoxic function of cells (75-77).
Although experiments have shown that iCART cells can maintain their tumoricidal function even after being exposed to inhibitory antigens, the possibility that some iCAR T cells will deteriorate due to repeated exposure to inhibitory antigens cannot be completely ruled out (75, 78). Moreover, since this well-designed regulation method is antigen-specific, it requires tissue-specific target antigens that are expressed by healthy tissues but are absent or down-regulated by tumor cells (75). Human leukocyte antigen (HLA) may be a suitable antigen with such characteristics, because it is expressed in all cell types, but is basically down-regulated by tumor cells, giving them the ability to evade T cell-mediated immune system responses (79 ).

Logic gate CART cell

A cross-signal CAR strategy has been developed in which the T cell activation signal and the costimulatory signal are separated from each other in two CARs with different antigen specificities, so that CART cells are equipped with a “dual or no” strategy (80–84 ). Conceptually, T cells are genetically modified to express two CARs. A receptor that only contains the CD3ζ signal domain and recognizes the target antigen with low affinity and the chimeric co-stimulatory receptor (CCR), which can recognize another target antigen with high affinity (80-84). In addition, the combination of CCR and antigen provides the costimulatory signal cascade necessary for T cell activation and effective cytotoxicity (80-84). Genetically modified T cells expressing these two constructs will not be effectively activated when they encounter normal cells. Due to insufficient activation signals, they only express one of the two antigens (Figure 3C) (80-84).

However, several issues raised questions about the practicality of the proposed strategy. Limitations, such as identifying two tumor antigens that are only expressed in a given type of cancer, and do not overlap in normal tissues (81). In addition, another limitation relates to the difficulty of designing CARs with a narrow range of optimal affinity or CARs that are actually suitable for almost a wide range of patients (80).

The results of in vitro studies have shown that CAR T cell signal transduction through counter-signaling of cells expressing only one TAA results in weaker cytokine secretion, and the secretion of cytokines when encountering tumor cells co-expressing two antigens It is also obvious (81). These findings indicate that the bispecific anti-signal CAR platform can enhance the therapeutic effect of CAR T cells on target cancer cells while reducing their cross-reactivity with normal tissues (81).


Γδ T cells that only costimulate CAR

Recently, researchers have used γδ T cells, which are a subset of T cells, in which TCR has γδ subunits instead of the more common αβ subunits (85, 86). γδ T cells account for approximately 1-10% of circulating T cells, but they are an important part of the immune system (87). Vγ9Vδ2 T cells are a subset of γδ T cells and have inherent tumor differentiation capabilities because they can recognize phosphoantigens of non-peptide tumor antigens and are a typical feature of metabolically dysregulated tumor cells (88). Researchers have studied a new strategy that uses Vγ9Vδ2T cells as the skeleton to generate a unique “costimulatory only domain CAR” (89). Unlike traditional αβ T cells (used as the main source for the production of CAR T cells), γδ T cells can recognize their target antigens without relying on MHC class I or II (90). Vγ9Vδ2TCR is the most common γδTCR expressed by γδ T cells (90). These TCRs recognize phosphoantigens, such as isopentenyl pyrophosphate (IPP), that are overproduced in cancer cells rather than healthy cells (90). γδ T cells distinguish cancer cells from normal cells by recognizing these antigens as “danger alerts” (90). Studies have shown that GD2-only co-stimulated CAR T cells produced from T cells and Vγ9Vδ2TCR are functional and only show a strong cytolytic response against GD2-positive neuroblastoma cells in vitro, while they are positive for GD2 Normal cells do not (see 89). This fact highlights the role of endogenous Vγ9Vδ2TCR, because CD3ζ signal is only provided by tumor cells that interact with endogenous Vγ9Vδ2TCR (Figure 4A) (89).

Strategies to deal with CAR T cell treatment obstacles Strategies to deal with CAR T cell treatment obstacles

In addition, other studies have also presented similar promising results, showing that CAR T cells (called γδCART cells) generated using Vδ2T cells can migrate to tumor cells and perform antigen cross-presentation (91). These findings indicate that γδCAR T cells can enter the tumor site and eliminate tumor cells, and at the same time take up the target antigen, resulting in the presentation of stimulating antigens to tumor infiltrating lymphocytes (TIL) through αβTCR (91). It has been proposed that tumors such as melanoma may be the right battlefield for these fighting cells because of their high tumor antigen frequency and large numbers of tumor-reactive lymphocytes and TIL (91). This fact can be considered as the advantage of γδCART cells over conventional CAR T cells, which may be very worthwhile in the treatment of solid tumors (91).
In short, it can still be concluded that γδ TCAR T cells may show promise for prospective clinical evaluation of solid tumors because they have unique and useful properties compared to conventional CAR T cells.


Universal CAR (UniCAR)

Another well-designed strategy to reduce the risk of extra-tumor side effects is to use a modular CAR platform called UniCAR. This strategy makes it possible to reversibly shut down the CAR system as quickly as possible in the event of adverse CAR T cell-mediated side effects (93). Conceptually, UniCAR consists of UniCAR effector T cells and engineered recombinant target modules that direct them to the surface of appropriate target cells (Figure 4B) (93). The specificity of the target module determines exactly which target cells UniCART cells should attack, and the safety index of quickly clearing them from the circulation is sufficient to prove that it switches UniCAR T cells to “on” and “off” (93). Since the antibody domains of UniCAR T cells target unique epitopes on the target module, they can build immune complexes when they are present (93). This will guide the transfer of UniCAR T cells to their target cells (93). On the other hand, in the absence of target modules, UniCAR T cells will automatically shut down, which makes their control more feasible than conventional CAR T cells (93-95). In order to minimize the risk of CRS during UniCAR T cell therapy, the target module should be quickly cleared from a low dose, and then adjusted and increased according to the appearance of unexpected side effects (93). Once the desired target cells are eliminated or any life-threatening adverse events occur, the termination of the targeted module administration will only result in the shutdown of UniCART cells (93).



4. Strategies to overcome control limitations after infusion

So far, various attempts have been made to control the activity of CAR T cells after they are injected into patients. This topic deserves special attention, because it can help control and prevent the previously mentioned CAR T cell-mediated toxicity, which can sometimes be life-threatening. In this section, we briefly outline the strategies aimed at controlling CAR expression on the surface of engineered T cells, as well as some of the most effective strategies developed to fully control CAR T cells after administration.


Lymphocyte specific protein tyrosine kinase (LCK) inhibition

It has been proven that the tyrosine kinase inhibitor dasatinib is an FDA-approved Philadelphia chromosome-positive chronic myeloid leukemia (CML) and ALL treatment drug. It inhibits LCK, thereby preventing the phosphorylation of CD3ζ and ZAP70 (96). Mestermann and colleagues have developed dasatinib to improve the safety index of CAR T cells (96). The mentioned mechanism can mediate the destruction of the downstream signaling cascade in CARs with CD28-CD3ζ or 4-1BB-CD3ζ activation modules (96). In addition, Dasatinib can induce rapid (3 hours) hibernation of CD8 and CD4-positive CAR T cells, which can last for several days without any negative impact on the viability of T cells (96). In addition, different dosing regimens of dasatinib can be used to partially or completely inhibit CAR T cell activity (96). Studies have shown that in the preclinical CRS mouse model, shortly after CAR T cell infusion, dasatinib can protect it from CRS, otherwise CRS may be lethal in a model that does not receive dasatinib (96). The main advantage of this method is that after the administration of dasatinib is interrupted, its inhibitory effect is quickly and completely reversed. Therefore, the previously affected CAR T cells can continue their normal signal transduction pathway and anti-tumor activity (96). The good pharmacodynamics of dasatinib is another advantage of this method, which allows the drug to be used multiple times so that the continuous “off” and “on” CAR T cell activity can be utilized. In conclusion, the administration of dasatinib in preclinical models and in vitro assays receiving CAR T cells can stop the cytolytic activity, cytokine production and expansion of CAR T cells, and can be used as a pharmacological prescribing of CAR T cells. Turn off the switch (96).



STOP CAR is a recently developed CAR platform composed of a recognition (R) chain responsible for antigen binding and a signal (S) chain responsible for T cell activation (98). The inner domains of these two different chains have a computationally designed protein pair that helps them dimerize into a functional heterodimer without the need for a dimerizer (98). This heterodimer is a chemically destructible heterodimer (CDH), which can be specifically destroyed and decomposed into two monomers by the administration of small molecules, such as A1331852 and A1155463 (they are Bcl-XL inhibitors). Body (98). Effective clinical applications, extended half-life and the availability of destructive small molecules with significant tolerance to humans are the principles for the design of this type of CDH (98). The basic purpose of the STOP CAR platform is to utilize spherical domains from modular proteins that do not interfere with T cell signaling near synapses (98).

Strategies to deal with CAR T cell treatment obstacles Strategies to deal with CAR T cell treatment obstacles



The activity of CAR T cells can be controlled (as a switch) by regulating the expression of CAR molecules on the surface of T cells. Recently, Juillerat et al. A CAR T cell activity control platform called T-OFF CARs (SWIFF-CARs) has been produced, which requires the use of protease-based small molecule assisted blocking (SMASh) (102). On this platform, the SWIFF-CAR construct consists of the CAR molecule, the subsequent protease cleavage site, the protease (HCV NS3 protease) and the degradation part called “degron” (102). In the absence of the cell-permeable protease inhibitor Asunaprevir, the protease cleaves its target site, causing the CAR to dissociate from the protease and degron (102). This change will cause the CAR molecule to shift to the cell surface, allowing it to have normal activity and emit a cascade signal (a state called “ON”) (102). On the other hand, in the presence of Asunaprevir, it binds to the binding site on the protease and inhibits its cleavage activity (102). Therefore, the CAR molecule will not dissociate from the protease cleavage site, and the protease and diaglon will cause proteolytic degradation of the CAR molecule (referred to as the “OFF” state) (Figure 5B) (102). This study shows that it is feasible to integrate a switch directly into the CAR structure, which can achieve reversible control of CAR surface expression (102).



5.  Suicide strategy

In the past few years, selective and permanent ablation of CAR T cells (including the occurrence of GVHD or on-target non-tumor toxicity) has been the subject of numerous studies. It has been recognized that a safety switch that can irreversibly eliminate CAR T cells during the aforementioned adverse events and the implementation of this strategy is an effective way to solve these challenges. One of these safety switches is based on suicide gene technology, which works through different mechanisms, such as metabolic pathways, reagent dimerization, and targeting by therapeutic mAbs. These switches will be discussed in detail in the following sections. It is worth mentioning that biological dormancy and good bioavailability and biodistribution characteristics are ideal characteristics of an ideal suicide switch activator (103).


Metabolic switch

The suicide switch can be based on the conversion of non-toxic compounds into toxic compounds, and ultimately play a role in killing the cells of the suicide switch. Unlike mammalian cell thymidine kinase, herpes simplex virus thymidine kinase (HSV-TK) has a very high affinity for ganciclovir (GCV), which is a nucleoside analog (104, 105). HSV-TK phosphorylates GCV into GCV-monophosphate (MP), and finally converts it into GCV-triphosphate (TP). DNA polymerase integrates GCV-TP into the leading strand of DNA, leading to GCV-induced chain termination (106, 107).

The HSV-TK/GCV suicide switch can also trigger the death-inducing signal cascade through the formation of Fas-related death domain protein (FADD) and the activation of caspase through ligand-independent CD95 aggregation (108). Although the HSV-TK switch has progressive effectiveness and potential immunogenicity risks (due to its viral origin), its benefit-risk ratio may still be clinically advantageous (Figure 6A) (104, 105).
Another example of this type of suicide-inducing switch involves cytosine deaminase (CD), which is a pyrimidine rescue enzyme (109). In terms of mechanism, 5-fluorouracil (5-FU) is the product of the antifungal drug 5-fluorocytosine (5-FC) deaminated by CD, so it acts as a highly cytotoxic compound that can induce cell death ( 109). In this regard, equipping CAR T cells with genes encoding enzymes such as HSV-TK or CD can irreversibly eliminate the infused CAR T cells in the event of adverse complications. In addition, the type 1 HSV-TK gene is also called a positron emission tomography (PET) reporter gene, which can be used to provide insights about CAR T cell transport to tumor sites (110).


MAb-based switch

Another suicide conversion strategy for selective in vivo ablation of CART cells is its genetic engineering to coordinate the expression of CAR and recombinant cell surface proteins (111-113). The recombinant protein should retain a complete conformational binding epitope recognized by a given pharmaceutical grade monoclonal antibody, such as cetuximab (EGFR specific mAb) or rituximab (CD20 specific mAb) (111 -113). This method makes the aforementioned CAR T cells susceptible to antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) when exposed to the agent, without changing their cytotoxic function (Figure 6B) (111-113).

It is also encouraging that no EGFRt molecule (a truncated form of the epidermal growth factor receptor) targeting EGFRt-positive CAR T cells showed signs of immunogenicity in preclinical mouse models (113). These findings support the hypothesis that the administration of cetuximab may lead to the long-term persistence of CART cells that undergo CD19-based CAR T cell therapy and undergo CD19 redirection, and the B cell area of ​​patients with poor B cell dysplasia and complete degeneration The room recovered and the tumor subsided (113). However, because mAb-based switches may severely damage healthy tissues expressing natural forms of recombinant proteins, there are still concerns about the clinical application of mAb-based switches (103).



Another example of an inducible safety switch is based on the recombinant fusion of modified FKBP12 (human FK506 binding protein) with the membrane-anchored intracellular domain of human caspase 9 or Fas (117, 118). This method allows for dimerization (117, 118) at will in the presence of a biologically inert dimerizer (such as AP1903). Conceptually, modified FKBP12 binds to synthetic dimerization drugs with high affinity, thereby inducing caspase 9 (iCasp9) or Fas-based suicide switches (117, 118) to undergo dimerization and subsequent activation. This leads to a caspase cascade, leading to apoptosis in cells expressing these constructs (Figure 6C) (117, 118). In addition, the study reported that within 30 minutes after a single dose of dimerization drugs in GVHD patients, >90% of iCasp9-equipped T cells were eliminated (118). In addition, this rapid elimination is related to the resolution of GVHD without recurrence (118). In addition, compared with other safety switches, the iCasp9 switch has some advantages, such as its low immunogenicity (due to the human origin of the iCasp9 suicide gene) and the use of biologically inert small molecules to activate it (rather than antiviral). Lovir and other drugs (118). These advantages make this safety switch a more suitable choice in the field of cell therapy. Moreover, through the involvement of the endogenous apoptotic pathway in the cell (within a few minutes after the administration of the dimerization drug), this switch mediates rapid cell death than other safety switches that require interference with DNA synthesis to induce cell death Much faster (119–124).



6. Strategies to overcome tumor recurrence

Due to the heterogeneity of antigens within a single tumor and between different patients, none of the antigens can be considered universal. Loss of antigen, down-regulation of antigen, or appearance of alternately spliced ​​antigens (which are no longer targeted by CAR T cells due to the loss of recognized epitopes) are all carefully designed antigen-dependent strategies executed by tumor cells to avoid immune recognition ( 125–127). Therefore, this phenomenon limits the tumor efficacy of targeted immunotherapy and leads to adverse clinical responses (125-127). Simultaneous multispecific targeting is one of the proposed strategies aimed at counteracting persistent tumor antigen escape variants that may provide enhanced immunotherapy-mediated relief. Compared with conventional CAR T cells, CAR T cells (Tandem CAR or TanCAR) whose CAR constructs are equipped with bispecific targeting domains in tandem or co-express two different chimeric receptors specific for two different TAAs. Somatic T cells may have advantages. These genetically manipulated T cells have the cytotoxic ability to target tumor cells that simultaneously express one or two antigens. Such CAR T cells have shown enhanced anti-tumor activity in vitro and in animal models of human tumors such as glioblastoma and B-cell malignancies (128-134).

According to the latest report from a clinical trial (NCT03185494), the trial evaluated the killing effect of bispecific CD19/CD22 redirected CAR T cells in adult R/R B-ALL patients. All 6 patients (100% ) All patients with MRD-negative CR had no episodes of neurotoxicity (135).

In addition, in the case of disease recurrence, UniCAR T cells may also be beneficial because after the introduction of a targeting module that targets a new tumor antigen (rather than a spliced ​​or expression deletion or expression deletion targeting module), UniCAR T cells Cells can trigger down-regulation of cytotoxic responses to evading tumor cells) (93). This ability proves why the CAR platform can be universally applied to different target antigens without the need to redesign new CAR constructs.



7.  Overcome immunosuppression

TME’s strategy

The TME nature of hypoxia can exploit the main differences in the nature of normal tissues and cancerous tissues to develop intelligent TME response or avoidance treatment methods. Low nutritional level, low extracellular pH (acidosis) and low oxygenation level (hypoxia) are the unique characteristics of TME (136, 137). The characteristic of the hypoxic microenvironment is that the oxygenation level is usually less than 1-2% (136,137). In addition, the immunosuppressive hypoxia-A2-adenosinegic axis is a very interesting feature of many treatment-resistant tumors (138). The discovery of the critical role of upstream factors in this pathway has led to the development of unique strategies to suppress the hypoxia/hypoxia inducible factor-1α (HIF-1α) axis (139,140). Preclinical studies on the A2A adenosine receptor (A2AR) and the adenosine-producing exonuclease CD73 have produced significant therapeutic effects (139-142).

The conclusion is that the hypoxic properties of TME mediate an increase in the level of anergy and exhaustion in T cells and CAR T cells, as well as a decrease in the production and secretion of cytokines (143). Researchers have shown that supplemental oxygenation and the use of oxygenation agents can reverse hypoxia in TME (143-145). They believe that this method can avoid the stabilization of HIF-1α and damage the hypoxic adenosinegic immunosuppressive axis (143-145). They demonstrated that this method can reprogram the properties of TME from “immune suppression” to “immune tolerance” (143-145). In addition, they also emphasized the clinical application of systemic oxygenation and oxygenation in combination with A2AR blockers to further address the immunosuppressive properties of TME (143). This strategy can destroy the upstream and downstream (hypoxia-HIF-1α and adenosine-A2AR) cascades of the immunosuppressive hypoxia-adenosine signal transduction axis, and can maximize the A2AR antagonist The treatment effect also increases the tumor’s sensitivity to cancer treatment (Figure 7) (143).

In addition, other researchers have also used the hypoxia of TME and designed intelligent self-decision making CAR T cells (146). They fused an oxygen-sensitive subdomain of HIF-1α to a CAR scaffold and generated CAR T cells that respond to hypoxia (146). This strategy has been developed to limit CAR expression to only those CAR T cells present in hypoxic TME (rather than CAR T cells in the non-hypoxic environment of non-malignant tissues) (146). Therefore, these CART cells can reduce the tumor-free effect of conventional CAR T cells (146).


Metabolic reprogramming of CAR T cells

The cellular metabolic conditions of T cells have a great influence on the effector function and differentiation status of T cells (147). Moreover, the components of CARs expressed in transduced T cells can affect their nutritional intake and metabolic status (147). The metabolic function relationships found in T cells can be used as tools to define their fate, activity, and effector functions (147). For example, studies have shown that the presence of the 4-1BB costimulatory domain in the CARs structure enables T cells to produce a central memory phenotype, enhances the oxidation and decomposition of fatty acids, and improves their expansion and durability (147). On the other hand, the CD28 costimulatory domain improves glycolysis and enables CAR T cells to develop an effect memory phenotype (147). In addition, supplementation such as L-arginine supplementation can balance the increase in arginine metabolism in activated T cells, while improving the tumor-killing function and inducing the development of the central memory phenotype (148).

A detailed understanding of the gene expression profiles of genes that are mainly involved in cell metabolism can help us achieve the goal of T cell metabolic reprogramming by changing the expression levels of metabolic genes. The proposed strategy is to overexpress the Akt pathway or Glut1 transporter to address this warning caused by leukemia cells (149). This strategy can restore the function of T cells to the level before tumor cells exerted a negative influence on them (149). In addition, PPAR-γ co-activator 1a, also known as PGC1a, is a transcription factor co-activator that affects a variety of cellular metabolic pathways. This metabolic regulator is down-regulated in T cells infiltrating the tumor site (150). Researchers have found that overexpression of PGC1a in T cells can rebuild their effector functions as well as their metabolism and mitochondrial activity (150).

In addition, research has used the characteristics and behavior of tumor cells and tissues to reprogram the metabolism of T cells (152, 153). In this regard, it has been found that necrotic tumor cells release potassium (K+) in TME, which leads to excessive accumulation of this ion (152). This phenomenon raises the intracellular concentration of K+ in tumor-infiltrating T cells to a level higher than the normal level, resulting in restriction of their nutritional intake (152). In addition, this accumulation in T cells down-regulates their protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signals and interferes with T cell activation signals (152). Researchers have shown that overexpressed K+ channels can reduce the increase in K+ content in cells, promote Akt/mTOR activity and restore the weakened T cell effector function (152). In summary, the above-mentioned metabolic reprogramming strategy either improves the response, activity and effector function of T cells and CAR T cells in TME, or avoids the negative impact of specific TME-specific modification of infiltrating T cells by tumor cells.


In short, these strategies may be applied in a synergistic manner to organize safer CAR T cell therapy while maximizing its tumor-killing efficacy. This is only good news for patients with difficult-to-treat malignant tumors.







(source:internet, reference only)

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