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Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy. Chimeric antigen receptor (CAR) T cell (CAR-T) therapy is revolutionary because it has produced a very effective and long-lasting clinical response.

CAR is an engineered synthetic receptor whose function is to redirect lymphocytes (most commonly T cells) to recognize and eliminate cells expressing specific target antigens.

The binding of CAR to the target antigen expressed on the cell surface does not depend on the MHC receptor, resulting in strong T cell activation and a strong anti-tumor response.

The unprecedented success of anti-CD19 CAR-T cell therapy in the treatment of B-cell malignancies led to its approval by the U.S. Food and Drug Administration (FDA) in 2017.

However, CAR-T cell therapy still has major limitations that must be addressed, including life-threatening CAR-T cell-related toxicity, limited efficacy on solid tumors, inhibition and resistance in B-cell malignancies, antigen escape, and limited Persistence, poor migration and tumor infiltration, and immunosuppressive microenvironment.

In addition, technicians must adapt to the needs of this growing and evolving field, for which it is necessary to develop educational programs to train them.

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy

Many methods have been proposed, including combining CAR-T cell therapy with other anti-cancer therapies, or adopting innovative CAR engineering strategies to improve anti-tumor efficacy, expand clinical efficacy, and limit toxicity. In this review article, Robert C. Sterner of the University of Wisconsin-Madison and Rosalie M. Sterner of the Mayo Clinic discussed the latest innovations in CAR-T cell engineering to improve the clinical efficacy of hematological malignancies and solid tumors , As well as strategies to overcome current limitations including antigen escape, CAR-T cell migration, tumor infiltration, immunosuppressive microenvironment and CAR-T cell-related toxicity (Figure 1).

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy
Figure 1. Limitations of CAR-T cell therapy. Picture from Blood Cancer Journal, 2021, doi:10.1038/s41408-021-00459-7.

CAR structure

CAR is a modular synthetic receptor, mainly composed of four parts:

(1) an extracellular target antigen-binding domain;

(2) a hinge region,

(3) A transmembrane domain (transmembrane domain);

(4) One or more intracellular signaling domains.

In this article, the two authors will discuss the basic principles of current CAR design.

(1) Antigen binding domain.

The antigen-binding domain is the part of the CAR that confers specificity to the target antigen. Historically, the antigen-binding domain was derived from the variable heavy chain (VH) and light chain (VL) of a monoclonal antibody. A flexible linker connects the VH and VL to form a single-chain variable fragment (scFv).

Generally speaking, the scFv present in the CAR targets cancer antigens on the extracellular surface, resulting in T cell activation independent of the major histocompatibility complex (MHC), despite the use of MHC-dependent simulated T cell receptor (TCR) ) CARs that recognize intracellular tumor-associated antigens have been described. In addition to simply recognizing and binding target epitopes, several features of scFv affect CAR function. For example, the interaction pattern between the VH and VL chains and the relative position of the complementarity determining region (CDR) affect the affinity and specificity of the CAR for its target epitope.

Affinity is a particularly important parameter of the antigen-binding domain, because it fundamentally determines the function of the CAR. In order to recognize antigens on the surface of tumor cells, induce CAR signal transduction and activate T cells, the antigen binding affinity of CAR must be high enough, but not high enough to cause CAR-T cells to undergo activation-induced cell death and cause toxicity. Although affinity is certainly one of the most important factors that further complicate the problem, studies have shown that even scFv with similar affinity can differentially affect CAR-T cell function.

Therefore, in order to optimize the binding of the CAR to the target antigen, other factors must be considered, such as epitope position, target antigen density, and avoid the use of scFv related to tonic signaling that is not related to ligand.

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy
Schematic diagram of the antigen binding site space, the picture is from FEBS Journal, 2011, doi:10.1111/j.1742-4658.2011.08207.x.

(2) Hinge area.

The hinge or spacer is defined as the area of extracellular structure that extends the binding unit from the transmembrane domain. The function of the hinge region is to provide flexibility to overcome space barriers and contribute to the length of the CAR to allow the antigen binding domain to contact the target epitope. Importantly, the selected hinge region seems to affect CAR function, because differences in the length and composition of the hinge region can affect flexibility, CAR expression, signal transduction, epitope recognition, intensity of activation output, and epitope recognition.

In addition to these effects, it has been proposed that the length of the hinge region is essential to provide sufficient intercellular distance to form an immune synapse. In principle, the “optimal” hinge region length depends on the target epitope position on the target cell and the level of spatial obstruction: the long hinge region provides increased flexibility and allows more effective access to the membrane proximal epitope or complex Glycosylated antigens, and short hinge regions more successfully bind to epitopes at the distal end of the membrane.

However, in practice, the appropriate hinge region length is often determined empirically and must be customized for each pair of specific antigen-binding domains. There are many examples in the literature, such as short-spaced CAR [CD19 and carcinoembryonic antigen (CEA)] and long-spaced CAR [mucin 1 (MUC1), receptor tyrosine kinase-like orphan receptor 1 (ROR1) membrane proximal gauge].

The most commonly used hinge regions are derived from the amino acid sequence of CD8, CD28, IgG1 or IgG4. However, the hinge region derived from IgG can cause CAR-T cell depletion, thereby reducing persistence in the body because they can interact with Fcγ receptors. These effects can be avoided by choosing different spacers or by further reforming the spacers based on functional or structural considerations.

(3) Transmembrane domain.

Among all the components of CAR, the transmembrane domain may be the least characterized region. The main function of the transmembrane domain is to anchor the CAR to the T cell membrane, although there is evidence that the transmembrane domain can also be related to CAR-T cell function. More specifically, studies have shown that the CAR transmembrane domain affects the expression level and stability of CAR, is active in the process of signal transduction or synapse formation, and dimerizes with endogenous signal molecules.

Most of the transmembrane domains are derived from natural proteins, including CD3ζ, CD4, CD8α or CD28. The effect of different transmembrane domains on CAR function has not been well studied, because the transmembrane domain is often changed according to the requirements of the extracellular compartment or intracellular signal transduction domain.

It is worth noting that the CD3ζ transmembrane domain may promote CAR-mediated T cell activation, because the CD3ζ transmembrane domain mediates CAR dimerization and integration into endogenous TCR. Compared with CAR with CD28 transmembrane domain, these beneficial effects of CD3ζ transmembrane domain are at the cost of reducing CAR stability.

The transmembrane domain and hinge region also seem to affect the cytokine production and activation-induced cell death (AICD) of CAR-T cells. This is because CARs with transmembrane domains and hinge regions derived from CD28 have CD8α The amount of TNF and IFNγ released by CAR-T cells in the transmembrane domain and hinge region is reduced, and the sensitivity to AICD is reduced. In general, studies have shown that connecting the proximal intracellular domain with the corresponding transmembrane domain may best promote the correct transmission of CAR-T cell signals, and the commonly used CD8α or CD28 transmembrane structure is used. The domain may enhance the expression and stability of CAR.

(4) Intracellular signal transduction domain.

It can be said that the most concern in CAR engineering is to understand the effect of CAR costimulation, and the goal is to generate CAR constructs with the best intracellular domains.

The first generation CAR designed in the late 1990s contained the CD3ζ or FcRγ signal transduction domain. Most CARs rely on CD3ζ-derived immunoreceptor tyrosine activation motifs to activate CAR-T cells.

However, an effective T cell response cannot be produced by signal transduction of these activation motifs alone. The durability of these first-generation CARs is not strong in vitro. These findings are echoed by clinical studies that have shown limited or no efficacy.

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy
CC49-ζ vector map, the picture is from J Immunother Cancer, 2017, doi:10.1186/s40425-017-0222-9.

Early in vivo models using B-cell malignancies confirmed the importance of costimulation in the persistence of CAR-T cells targeting CD-19. By adding a costimulatory domain, IL-2 production and cell proliferation after repeated antigen exposure are improved.

With the understanding of the importance of costimulation for persistent CAR-T cell therapy, people have constructed a second-generation CAR with a costimulatory domain in series with the signal transduction domain in CD3ζ cells. The two most common FDA-approved costimulatory domains, CD28 and 4-1BB (CD137), are associated with higher patient response rates.

The functions and metabolic profiles of these two co-stimulatory domains are different: CAR-T cells with CD28 domain differentiate into effector memory T cells, which mainly use aerobic glycolysis, while CAR-T cells with 4-1BB domain Differentiate into central memory T cells and show increased mitochondrial biogenesis and oxidative metabolism.

Clinically, second-generation CAR-T cells have produced strong therapeutic responses in some hematological malignancies, including chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma and multiple myeloma.

The efficacy of second-generation CAR-T cells in solid tumors is currently being studied, including glioblastoma, advanced sarcoma, liver metastases, mesothelioma, ovarian cancer, and pancreatic cancer. Several alternative costimulatory domains, such as inducible T cell costimulator (ICOS), CD27, MYD88, CD40, and OX40 (CD134) have shown preclinical efficacy, although clinical studies are still pending.

It is speculated that costimulation of only one domain produces incomplete activation, which leads to the production of the third-generation CAR. The third-generation CAR integrates two co-stimulatory domains in series with CD3ζ. Preclinical studies of the third-generation CAR have produced mixed results. Specifically, CARs integrated with CD28 and 4-1BB signals lead to stronger cytokine production in lymphomas.

Compared with second-generation CARs, they show better in vivo anti-tumor responses in lung metastases. In leukemia and pancreatic cancer models, the third-generation CAR did not show in vivo therapeutic advantages, and failed to outperform the second-generation CAR in their respective models.

Limitations of CAR-T cell therapy

(1) Antigen escape.

One of the most challenging limitations of CAR-T cell therapy is that tumors are resistant to single-antigen-targeted CAR (ie, single-antigen-targeted CAR) constructs. Although the initial single-antigen targeting CAR-T cells can provide a high response rate, in patients receiving these CAR-T cell therapy, a considerable number of patients’ malignant cells show partial or complete loss of target antigen expression.

This phenomenon is called antigen escape. For example, although 70% to 90% of relapsed and/or refractory ALL patients show a durable response to CD19-targeted CAR-T cell therapy, recent follow-up data indicate that there is a common disease resistance mechanism. Including 30% to 70% of patients with relapsed disease after treatment, down-regulation/loss of CD19 antigen.

Similarly, in patients with multiple myeloma who are receiving BCMA-targeted CAR-T cell therapy, down-regulation or loss of BCMA expression has been observed. A similar pattern of antigen escape resistance has been observed in solid tumors.

For example, case reports of CAR-T cell therapy targeting IL13Ra2 in glioblastoma showed that IL13Ra2 expression was reduced when the tumor recurred.

In order to reduce the recurrence rate of CAR-T cell therapy for hematological malignancies and solid tumors, many strategies now rely on targeting multiple antigens. These strategies all use dual CAR constructs or tandem CARs. Tandem CAR refers to a single CAR construct containing two scFvs in order to target multiple tumor target antigens at the same time. From a clinical point of view, these two strategies may lead to long-term and durable remission rates.

There have been multiple clinical trials targeting CD19 and CD20 or CD19 and CD22. Excitingly, the preliminary results of clinical trials using dual-targeted CAR-T cells (CD19/CD22 or CD19/BCMA) have shown promising results. More specifically, the preliminary clinical trial results of CD19/CD22 CAR-T cell therapy showed good efficacy in adult patients with ALL and diffuse large B-cell lymphoma.

In addition, the preliminary results of BCMA/CD19 targeting CAR-T cells in the treatment of multiple myeloma show that BCMA/CD19 targeting CAR has high efficacy and good safety. In solid tumors, several tandem CARs have been tested in preclinical models, including tandem CARs targeting HER2 and IL13Ra2 in glioblastoma and tandem CARs targeting HER2 and MUC1 in breast cancer.

In both cases, dual targeting resulted in superior anti-tumor responses compared to single-targeted therapy. In the study of glioblastoma, compared with the other two dual-targeted therapies, the tandem CAR targeting HER2 and IL13Ra2 resulted in improved anti-tumor activity and reduced antigen escape.

This study illustrates the importance of optimizing the selection of target antigens, which can not only improve the anti-tumor response, but also reduce antigen escape and prevent recurrence.

(2) On-target off-tumor effects

One of the challenges of targeting solid tumor antigens is that solid tumor antigens are usually also expressed at different levels in normal tissues. Therefore, antigen selection is very important in CAR design, not only to ensure the therapeutic effect, but also to limit off-target toxicity.

One potential way to overcome targeting solid tumor antigens that are also present in normal tissues is to target post-translational modifications that are limited to tumors, such as truncated O-glycans overexpressed in solid tumors, such as Tn(GalNAca1-O-Ser/ Thr) and sialyl-Tn (STn) (NeuAca2–6-GalNAca1-O-Ser/Thr).

Four major CAR-T cell targets have been studied, including TAG7228, B7-H3, MUC1 and MUC16. Although the first generation of CAR-T cells targeting TAG72 in colorectal cancer did not produce an anti-tumor response, a new version of the second generation of TAG72-CAR-T cells and other tumor-restricted post-translational modifications are currently being studied.

In order to expand the clinical application of CAR-T cell therapy in hematological malignancies and solid tumors, it will be necessary to further develop innovative strategies to reduce antigen escape and select antigens that can induce sufficient anti-tumor efficacy while minimizing toxic side effects. .

CAR-T cells targeting B7-H3, the picture is from Cancer Cell, 2019, doi:10.1016/j.ccell.2019.01.002.

(3) The migration and tumor infiltration of CAR-T cells.

Compared with hematological malignancies, CAR-T cell therapy for solid tumors is limited by the ability of CAR-T cells to migrate and infiltrate into solid tumors because of the immunosuppressive tumor microenvironment and physical tumor barriers, such as tumor stroma.

The permeation and migration of CAR-T cells are improved. One strategy to improve these limitations is to use delivery routes other than systemic delivery. This is because local delivery (1) eliminates the need for CAR-T cells to migrate to the disease site, and (2) limits off-target toxicity. The target activity of CAR-T cells is directed against tumor cells, thereby minimizing their interaction with normal tissues.

Preclinical models have proven that intravenous injection of CAR-T cells targeting HER2 and CAR-T cells targeting IL13Ra2 has excellent therapeutic effects in breast cancer brain metastases and glioblastoma. These studies have led to three ongoing clinical trials. These three clinical trials are targeted at glioblastoma (NCT02208362, NCT03389230) and recurrent brain or leptomeningeal metastasis (NCT03696030) for intravenous injection of CAR-T cells.

Similarly, preclinical models have shown the superiority of intrapleural injection of CAR-T cells in the treatment of malignant pleural mesothelioma, which has led to an ongoing phase 1 clinical trial (NCT02414269). Although local injection seems to have a better effect, in theory this method is limited to single tumor lesions/oligometastatic disease.

A recently developed strategy that appears to significantly improve the migration of CAR-T cells involves expressing chemokine receptors on the surface of CAR-T cells to match and respond to tumor-derived chemokines. For example, recent studies have shown that genetically modified αvβ6-CAR-T cells expressing CXCR2 integrin or CAR-T cells overexpressing CXCR1 or CXCR2 both enhance migration and significantly improve anti-tumor efficacy.

Physical barriers such as tumor stroma also limit CAR-T cell therapy because these physical barriers prevent the penetration of tumors. Tumor stroma is mainly composed of extracellular matrix, and in the extracellular matrix, heparan sulfate proteoglycan (HSPG) is the main component that CAR-T cells must degrade to enter the tumor.

CAR-T cells expressing heparanase that degrade HSPG after genetic modification showed enhanced tumor invasion and anti-tumor activity. Similarly, CAR-T cells targeting fibroblast activation protein (FAP) exhibit increased cytotoxicity by reducing tumor fibroblasts in animal models.

In the future, it is necessary to develop innovative delivery strategies and methods to increase tumor penetration in order to extend the therapeutic effect to complex solid tumors and metastases.

(4) Immunosuppressive microenvironment.

In the tumor microenvironment, many cell types that drive immunosuppression can infiltrate solid tumors, including bone marrow-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Treg).

These tumor-infiltrating cells drive tumors to promote the production of cytokines, chemokines, and growth factors. In addition, immune checkpoint pathways, such as PD-1 or CTLA-4, can be used to reduce anti-tumor immunity. One of the main reasons for the lack of response or weak response to CAR-T cell therapy is poor T cell expansion and short-term persistence of T cells.

It is speculated that this T cell failure is triggered by a common inhibitory pathway. Therefore, the combined immunotherapy of CAR-T cells and immune checkpoint blockade is considered to be the next frontier of immunotherapy, because it provides two elements necessary for a strong immune response: (1) CAR-T cells that infiltrate tumors (2) PD-1/PD-L1 blockade can ensure the persistence and function of T cells.

In hematological malignancies, in a single-center study at Penn Children’s Hospital, 14 B-ALL children who had received a large number of treatments in advance were treated with PD-1 blockade and CD19 CAR-T cell combination therapy, which improved CAR- Persistence of T cells and achieved better clinical results. In solid tumors, there are currently many studies aimed at evaluating the response rate of this type of combination therapy.

In an intriguing study, 11 patients with mesothelioma received a single dose of mesothelin targeting CAR-T cells and at least three doses of anti-PD-1 drugs after pretreatment with cyclophosphamide. The result was 72 % Response rate and complete metabolic response of two patients. Combining other forms of immunotherapy strategies may still be necessary in order to counter the inhibitory signals present in the tumor microenvironment.

Recent research work has focused on genetic modification of CAR that is resistant to immunosuppressive factors (such as TGF β-mediated inhibitory signals) in the unfriendly tumor microenvironment. Another interesting strategy involves genetic modification of CAR-T cells to provide immunostimulatory signals in the form of stimulatory cytokines, increase the survival, proliferation, and anti-tumor activity of T cells, and rebalance the tumor microenvironment.

Many studies have explored many cytokines to create these “armored CARs.” Research that focuses on the expression of pro-inflammatory cytokines rather than on inhibitory signals relies on IL-12 secretion, IL-15 expression, and redirection of immunosuppressive cytokine (such as IL-4) signals to pro-inflammatory cytokines.

Although immune checkpoint blocking-CAR-T cell combination therapy is likely to be a new immunotherapy option, it must be recognized that even this combination may still be insufficient to induce T cell infiltration and effector functions. Therefore, additional research that combines CAR-T cell therapy and immune checkpoint blockade with other immunotherapies/strategies may be necessary to achieve T cell infiltration and effector functions in complex hematological malignancies or solid tumors.

(5) CAR-T cell-related toxicity. Although CAR-T cell therapy is already a revolutionary cancer treatment tool, the high rate of side effects and some deaths prevent CAR-T cell therapy from becoming a first-line treatment.

The key factors that may determine the incidence and severity of cytokine release syndrome (CRS), hematopoietic syndrome (HLH)/macrophage activation syndrome (MAS) and/or immune effector cell-related neurotoxic syndrome (ICANS) It is the design of CAR, specific target and tumor type. So far, the toxicity of CAR-T cell therapy has been most widely described in patients who received the first CAR-T cell therapy approved by the FDA—CAR-T cells that target CD19.

Even in clinical trials with the highest response rate, patients can experience serious life-threatening events. Specifically, in patients with acute lymphoblastic leukemia/lymphoma (ALL/LBL) receiving CAR-T cell therapy, almost all patients have at least some less severe toxic manifestations, while 23% to 46% The patient showed severe hyperphysiological cytokine production and a large amount of T cell proliferation in the body.

These toxic levels of systemic cytokine release and severe immune cell cross-activation in some patients lead to the following toxicities:

1) CRS, which is related to the production of superphysiological cytokines and the proliferation of a large number of T cells in the body;

2) HLH/MAS It is defined as a severe hyperinflammatory syndrome characterized by CRS, elevated serum ferritin and hemophagocytosis, renal failure, liver enzymes, splenomegaly, pulmonary edema and/or natural killer (NK) Loss of cell activity;

3) ICANS, which is characterized by elevated levels of cerebrospinal fluid cytokines and destruction of the blood-brain barrier.

From a mechanism point of view, CRS is due to the extensive activation of the delivered CAR-T cells, resulting in the release of a large number of cytokines. The clinical manifestations of mild CRS are fever, accompanied by fatigue, diarrhea, headache, skin rash, arthralgia, and myalgia. In more severe cases, patients may experience hypotension, cardiac dysfunction, circulatory failure, respiratory failure, and renal failure.

Functional failure, multiple organ system failure, and may progress to death. In general, 77%-93% of leukemia patients receiving CAR-T cell therapy and 37%-93% of lymphoma patients receiving CAR-T cell therapy have any grade of CRS, and tisagenlecleucel is used to treat recurrence 46% of patients with refractory B-ALL and 13%-18% of patients treated with axicabtagene ciloleucel and tisagenlecleucel for diffuse large B-cell lymphoma showed ≥ grade 3 CRS.

In terms of pathophysiology, CRS is considered to be mainly mediated by IL-6. Therefore, CRS management relies on the use of tocilizumab and corticosteroids to block IL-6 receptors. Even with the use of tocilizumab, which is approved by the FDA for the treatment of severe CRS, severe CRS and death still occur.

Interestingly, HLH/MAS after CAR-T cell therapy is resistant to IL-6 inhibition and may require chemotherapy. Although the incidence of HLH/MAS after CAR-T cell therapy is unclear due to overlap with high-grade CRS, it has been reported in about 1% of patients receiving CAR-T cell therapy. In the case of neurotoxicity, the underlying pathophysiology and mechanism are not fully understood.

The clinical manifestations of ICANS range from confusion, headaches, attention disorders, difficulty finding words, focal neurological dysfunction or encephalopathy to life-threatening cerebral edema, transient coma, or seizures. Neurotoxicity after CAR-T cell therapy is relatively common. Among the leukemia patients and lymphoma patients undergoing treatment, up to 67% and 62% of patients have neurotoxicity, respectively.

The management of neurotoxicity focuses on corticosteroids, because IL-6 inhibitors are usually ineffective against neurotoxicity associated with CAR-T cell therapy. So far, there are still no approved therapies to prevent the above toxicity, which makes it important to optimize CAR design and adopt other strategies to reduce CAR-induced toxicity. Below, these authors review the lessons learned in CAR design to reduce toxicity, as well as additional strategies adopted to alleviate the toxicity in CAR-T cell therapy.

Countermeasures against the limitations of CAR-T cells

In order to achieve an effective therapeutic response, the antigen-binding domain of CAR-T cells must bind to its target epitope and reach a minimum threshold level to induce CAR-T cell activation and cytokine secretion. But at the same time, there is also a certain activation threshold level.

When the threshold level is exceeded, cytokines and immune system activation at toxic levels will be produced. In other words, CAR-T cells must remain within their treatment window to exert clinical efficacy, because exceeding the treatment window will lead to toxicity.

From the perspective of genetic modification, the activation degree and activation kinetics of CAR-T cells are affected by several factors, including but not limited to the level of tumor antigens expressed on the surface of malignant cells, tumor burden, and antigen binding domains. The affinity of the target epitope, and the costimulatory domain of the CAR.

Therefore, it is necessary to carefully consider several components of CAR’s modular structure in terms of optimizing therapeutic effects and limiting toxicity.

(1) Change the CAR structure.

One way to reduce toxicity is by changing the affinity of the antigen-binding domain of CAR-T cells. Decreasing the affinity of the antigen-binding domain is expected to lead to an increased requirement for higher antigen density on the surface of tumor cells to achieve high levels of activation.

Therefore, it is expected that the decreased antigen affinity will circumvent the targeting of healthy tissues with relatively low amounts of antigen. Studies exploring this principle have shown that, compared with antigen-binding domains with low nanomolar/sub-nanomolar affinity, antigen-binding domains with micromolar affinity are more selective for tumors with higher levels of target antigen expression. Much higher.

It is also possible to modulate the secretion of cytokines by modifying the hinge region and transmembrane region of activated CAR-T cells.

For example, in CARs targeting CD19, modification of the amino acid sequence of the hinge region and transmembrane region derived from CD8-α can lead to a decrease in the release of cytokines and a decrease in the proliferation of CAR-T cells. Optimizing the hinge region and transmembrane region may be a useful method to reduce toxicity, because in phase 1 clinical trials, these CARs modified in the hinge region and transmembrane region caused 54.5% of B-cell lymphoma patients (6/11 Patients) complete remission, and importantly, no CRS or ICANS events of grade >1 occurred.

The costimulatory domain provides another modifiable area in CAR design, which can be customized according to tumor type, tumor burden, antigen density, target antigen-antigen binding domain pairs, and potential toxic side effects. Specifically, the 4-1BB domain leads to a lower risk of toxicity, higher T cell durability, and a lower peak level of T cell proliferation, while the CD28 costimulatory domain is related to CAR-T cell activity.

The onset and subsequent failure of the disease is more rapid. Therefore, the costimulatory domain of 4-1BB, which produces less toxicity, may be particularly useful in the case of tumors with high disease burden and/or high antigen density, while the costimulatory domain of CD28 may be in the lower total surface antigen density and/ Or in the case of a CAR with a low-affinity antigen-binding domain, it is required to reach the required T cell activation threshold.

(2) Reduce immunogenicity.

The host immune system’s recognition of CAR constructs may promote cytokine-related toxicity. Therefore, it may be advantageous to use human or humanized antibody fragments instead of murine CARs to reduce the immunogenicity of CARs. In addition, the hinge region and/or transmembrane domain can be modified to reduce the immunogenicity of the CAR, and interestingly, the persistence of CAR-T cells is improved.

(3) Modify CAR-transduced T cells to reduce neurotoxicity.

An exciting recently developed approach to prevent CAR-T cell cytokine toxicity is based on the modification of CAR-transduced T cells. Cytokines and myeloid cells seem to play an important role in CAR-T cell-induced neurotoxicity, because there are reports that CD14 cells are significantly increased in patients with grade 3 or higher neurotoxicity, and a key target CAR-T cell clinical trials of B-cell lymphoma have shown that among the serum biomarkers associated with neurotoxicity of grade 3 or higher, the increase in GM-CSF is most significantly associated with neurotoxicity. Recent preclinical studies have shown that after lenzilumab is used to inhibit the cytokine GM-CSF that activates macrophages and monocytes, neurotoxicity and CRS are reduced, and CAR-T cell activity is increased. Mutation inactivation of GM-CSF seems to have a similar effect in CAR-transduced T cells.

Therefore, these findings indicate that GM-CSF neutralization helps reduce neurotoxicity and reduce CRS. In addition, removing tyrosine hydroxylase in a myeloid cell-specific manner or using methyltyrosine to inhibit this enzyme leads to a decrease in catecholamine and cytokine levels.

Preclinical evidence also shows that IL-1 receptor antagonists reduce a form of neuroinflammation in a mouse model of leukemia/lymphoma treated with CAR-T cells targeting CD19.

(4) CAR “close switch”.

Another potential way to alleviate CAR-T cytotoxicity is through the implementation of “switch off” or suicide gene strategies. Such a strategy will help to selectively reduce genetically modified cells through secondary inducer treatment when adverse events occur.

Several methods using these concepts have been developed. For example, CAR constructs that independently express full-length CD20 or CD20 mimotopes or are genetically modified to express full-length CD20 or CD20 mimotopes promote the elimination of CAR-T cells by rituximab treatment.

However, a limitation of this method is the relatively slow occurrence of antibody-mediated CAR-T cell elimination, which may limit the need for this method to immediately reverse this toxicity during severe acute cytokine-mediated toxicity. Efficacy in patients.

This prompted the development of faster switches, such as inductive cas9. In a clinical trial, inducible cas9 eliminated more than 90% of genetically modified T cells within 30 minutes.

Other strategies rely on protease-based small molecule assisted shutdown CAR (SMASh-CAR), which is also known as switch-off CAR (SWIFF-CAR). The biggest limitation of suicide strategies or other similar methods is that, although they are attractive to ensure safety, their use will suddenly stop the treatment of rapidly progressing diseases.

This restriction has become a powerful motivation for people to develop strategies to ensure safety, and suicide gene activation is the last resort. One method with exciting potential involves the use of the tyrosine kinase inhibitor dasatinib, whose function is to inhibit T cell activation by inhibiting the proximal TCR signal kinase.

In the preclinical model, dasatinib quickly and reversibly prevented CAR-T cell activation, and the result of dasatinib administered early after CAR-T cell infusion significantly reduced the occurrence of lethal CRS in mice mortality rate.

Therefore, this approach seems to provide a temporary inhibition of CAR-T cell function and may allow CAR-T cell therapy to be rescued after the toxicity subsides.

In the future, the development of additional innovative methods that temporarily inhibit the function of CAR-T cells and allow CAR-T cell therapy to rescue after the toxicity subsides will be a necessary condition for CAR-T cell therapy to move towards the first-line treatment of hematological malignancies and solid tumors.

Conclusion

CAR is a modular synthetic receptor, mainly composed of four parts: an extracellular target antigen binding domain, a hinge region, a transmembrane domain, and one or more intracellular signal transduction domains.

CAR-T cells have completely changed the treatment of certain hematological malignancies. However, there are still obstacles. Training technicians to meet the needs of this complex and evolving field is challenging and requires the development of innovative courses. Antigen selection is critical to CAR-T cell function.

Due to the selective pressure of CAR-T cells, tumor cells can down-regulate antigen expression. Even with proper antigen targeting, off-target effects will occur and cause related toxicity. In solid tumors, allowing CAR-T cells to migrate to and infiltrate the tumor is a major challenge. The immunosuppressive microenvironment of malignant tumors can exacerbate this obstacle.

Effective treatment can also lead to the risk of CAR-T cell-related toxicity (such as CRS and neurotoxicity). However, despite the challenges, new strategies and potential solutions are still evolving and may provide a path for more effective and safer treatments in the future.

(source:internet, reference only)

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Current Limitations and Potential Strategies of CAR-T Cell Therapy

Nature : Current Limitations and Potential Strategies of CAR-T Cell Therapy