December 4, 2021

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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

Nature: Current limitations and potential strategies of CAR-T cell therapy

Nature: Current limitations and potential strategies of CAR-T cell therapy.  Introduce a recent article on nature, mainly discussing the latest innovations in CAR-T cell engineering technology to improve the clinical efficacy of hematological malignancies and solid tumors, as well as strategies to overcome current limitations (Table 1), including antigens Escape, CAR-T cell trafficking, tumor infiltration, immunosuppressive microenvironment and CAR-T cell-related toxicity (Figure 1).

Nature: Current limitations and potential strategies of CAR-T cell therapy Nature: Current limitations and potential strategies of CAR-T cell therapy


Limitations of CAR-T cell therapy

Antigen escape

One of the most challenging limitations of CAR-T cell therapy is the development of tumor resistance to CAR constructs that target a single antigen. Although CAR-T cells that initially target a single antigen can provide a high response rate, the malignant cells of most patients treated with these CAR-T cells show partial or complete loss of target antigen expression. This phenomenon is called antigen escape.

For example, although 70-90% of patients with relapsed and/or refractory ALL show a durable response to CD19-targeted CAR-T cell therapy, recent follow-up data indicate that a common anti-disease mechanism has been developed , Including the down-regulation/loss of CD19 antigen, can be observed in 30–70% of patients with relapsed disease after treatment [42,43]. Similarly, the down-regulation or loss of BCMA expression has been observed in multiple myeloma patients receiving BCM-targeted CAR-T cell therapy [44,45,46]. A similar pattern of antigen escape resistance has been observed in solid tumors. For example, a CAR-T cell therapy case report targeting IL13Ra2 in glioblastoma showed that tumor recurrence showed decreased IL13Ra2 expression [47].

In order to reduce the recurrence rate of hematological malignancies and solid tumors in CAR-T cell therapy, many strategies now rely on targeting multiple antigens. These use dual CAR constructs or the use of tandem CARs, which are a single CAR construct containing two scFvs in order to target multiple target tumor antigens at the same time. From a clinical point of view, these two strategies may lead to a prolonged and lasting remission rate, and there are several CD19 and CD20 or CD 19 and CD22 clinical trials [25].

It is exciting that the preliminary results of clinical trials using dual-target CAR-T cells (CD19/CD22 or CD19/BCMA) have shown encouraging results [48,49,50,51]. More specifically, the results of preliminary clinical trials of CD19/CD22 CAR-T cell therapy indicate that the therapy has promising prospects in adult patients with ALL and diffuse large B-cell lymphoma [50,51]. In addition, the preliminary results of CAR with BCMA/CD19 as the target in the treatment of multiple myeloma show that CAR with BCMA/CD19 as the target has high safety and good safety [48,49].

In solid tumors, multiple tandem CARs have been tested in preclinical models, including HER2 and IL13Ra2 in glioblastoma and HER2 and MUC1 in breast cancer. In both cases, dual targeted therapy produces better anti-tumor response than single targeted therapy [28,52]. In the study of glioblastoma, CARs targeting HER2 and IL13Ra2 improved anti-tumor activity and reduced antigen escape compared with the other two dual-targeted therapies [53]. This study shows the importance of optimizing the choice of target antigen, not only can improve the anti-tumor response, but also can reduce the antigen escape mechanism to prevent recurrence.

Non-tumor effect on target

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

A potential way to overcome antigen targeting solid tumors that also exist on normal tissues is to target tumor-restricted post-translational modifications, such as truncated O-glycans overexpressed by solid tumors, such as Tn (GalNAca1-O-Ser/Thr ) And sialic acid Tn (STn) (NeuAca2-6-6GalNAca1-O-Ser/Thr) [54]. Four major CAR-T cell targets have been studied, including TAG7228, B7-H3[55,56], MUC1[16] and MUC16[57.58].

Although the first-generation CAR-T cells targeting TAG72 did not produce anti-tumor responses in colorectal cancer, a new version of the second-generation TAG72-CAR-T cells and other tumor-restricted post-translational modifications are currently being studied [28, 59].

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


CAR-T cell transport and tumor infiltration

Compared with hematological malignancies, solid tumor CAR-T cell therapy is limited by the ability of CAR-T cells to transport and infiltrate solid tumors, because the immunosuppressive tumor microenvironment and physical tumor barriers (such as tumor stroma) restrict CAR The penetration and migration of T cells. One strategy to improve these limitations is to use other routes of administration besides systemic administration, because local administration (1) eliminates the need to transport CAR-T cells to the disease site, and (2) limits the target. In addition to tumor toxicity, because the targeting activity of CAR-T cells is aimed at tumor cells, it can minimize the interaction with normal tissues [60].

Preclinical models have proved that the intraventricular injection of CAR-T cells targeting HER2[61] and IL13Ra2[62] for breast cancer brain metastasis and glioblastoma has superior therapeutic effects. These studies have three ongoing clinical trials, investigating CAR-T cells (NCT02208362, NCT03389230) and recurrent brain or pial metastases (NCT03696030) in intraventricular injection of glioblastoma.

Similarly, the preclinical model showed that the CAR-T cell therapy for malignant pleural mesothelioma by intrapleural injection was better, which led to the ongoing phase 1 clinical trial (NCT02414269) [63]. Although local injection seems to have a higher efficacy, theoretically, this method is limited to single tumor lesions/less metastatic diseases [25].

A recently developed strategy that seems to significantly improve CAR-T cell trafficking involves expressing chemokine receptors on CAR-T cells that match and respond to tumor-derived chemokines [64]. For example, recent studies have shown that integrin αvβ6-CAR-T cells can be modified to express CXCR2 or CAR-T cells overexpressing CXCR1 or CXCR2, which can not only enhance the transport efficiency, but also significantly improve the anti-tumor efficacy [64,65 ,66]. Physical barriers such as tumor stroma also limit CAR-T cell therapy because these physical barriers prevent tumor penetration.

The matrix is ​​mainly composed of extracellular matrix, in which heparan sulfate proteoglycan (HSPG) is the main component of CAR-T cells that must be degraded to enter the tumor [67]. CAR-T cells engineered to express heparanase, an enzyme that degrades HSPG, show enhanced tumor invasion and anti-tumor activity [68]. Similarly, CAR-T cells targeting fibroblast activation protein (FAP) showed enhanced cytotoxicity by reducing tumor fibroblasts in animal models [69]. In the future, innovative delivery strategies and methods need to be developed to improve tumor penetration in order to extend the therapeutic efficacy to complex solid tumors and metastases.


Immunosuppressive microenvironment

In the tumor microenvironment, many cell types that drive immunosuppression can infiltrate solid tumors, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Tregs) [70]. These infiltrates and tumor cells drive the production of cytokines, chemokines and growth factors that promote tumors.

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 T cell persistence. It is speculated that the development of this T cell exhaustion is triggered by a common inhibitory pathway.

Therefore, the combination immunotherapy that combines CAR-T cells with checkpoint block is considered to be the next frontier of immunotherapy because it provides two elements necessary for a strong immune response: 1 CAR-T cells, which provide Infiltration; 2 PD-1/PD-L1 blockade can ensure continuous T cell persistence and function [72].

In hematological malignancies, in a single-center study conducted at the Pennsylvania Children’s Hospital, PD-1 blockers and CD19 CAR-T cell combination therapy treated 14 severely preconditioned B-ALL children and improved Improved the persistence of CAR-T cells and improved the outcome. In solid tumors, there are currently many studies aimed at evaluating the response rate of combination therapy [71,74].

A fascinating study in which 11 mesothelioma patients who received cyclophosphamide pretreatment, followed by a single dose of mesothelin-targeted CAR-T cells and at least three doses of anti-PD-1 agents, made two patients The reaction rate reached 72%, and the metabolic reaction was completed [75]. In order to combat the inhibitory signals present in the tumor microenvironment, it may still be necessary to combine other forms of immunotherapy.

Recent efforts have focused on engineered CARs that are resistant to immunosuppressive factors in the hostile tumor microenvironment, such as TGFβ-mediated inhibitory signals [76]. Another fascinating strategy involves modifying CAR-T cells to provide immunostimulatory signals in the form of stimulatory cytokines, thereby increasing T cell survival, proliferation, anti-tumor activity and rebalancing the tumor microenvironment [77]. Many studies have studied many cytokines to create these “armored CARs.”

Research that focuses on the expression of pro-inflammatory cytokines rather than suppressing signals relies on IL-12 secretion [78], IL-15 expression [79], and the signal transduction of immunosuppressive cytokines (such as IL-4) to pro-inflammatory Cytokines [80].

Although combined checkpoint blocking CAR-T cell therapy may be a new immunotherapy, it is important to realize that even this combination therapy may not be sufficient to induce T cell infiltration and effector function. Therefore, it may be necessary to conduct other research to combine CAR-T cell therapy and checkpoint blockade with other immunotherapies/strategies to cause T cell infiltration and effector functions in complex hematological malignancies or solid tumors.


CAR-T cell-related toxicity

Although CAR-T cell therapy has become a revolutionary cancer treatment tool, its high toxicity and certain lethality have made CAR-T cell therapy a first-line treatment. The key factors that may determine the incidence and severity of CRS, HLH/MAS and/or ICANS are CAR design, specific target and tumor type [81].

So far, the potential toxicity of CAR-T cell therapy has been the most widely characterized in patients who received the first CAR-T cell therapy approved by the FDA, CD19-directed CARs[82,83]. Even in clinical trials with the highest response rate, serious, life-threatening events have occurred among patients [4,5,84]. In particular, in the case of acute lymphocytic leukemia/lymphoma (ALL/LBL) patients treated with CAR-T cell therapy, almost all patients have at least mild toxicity, and 23-46% of patients have severe toxicity. The production of superphysiological cytokines and the expansion of large numbers of T cells in vivo [85].

In some patients, these toxic levels of systemic cytokine release and severe immune cell cross-activation lead to the following toxicities: 1. Cytokine release syndrome (CRS), which is related to the production of superphysiological cytokines and the expansion of a large number of T cells in the body; 2. Hemophagocytic lymphohistiocytosis and/or macrophage activation syndrome (MAS), defined as severe hyperinflammatory syndrome, characterized by CRS and elevated serum ferritin and hemophagocytosis, renal failure, liver enzymes , Splenomegaly, pulmonary edema and/or lack of NK cell activity, and 3 immune effector cell combination-related neurotoxic syndrome (ICANS), which is characterized by elevated levels of cerebrospinal fluid cytokines and destruction of the blood-brain barrier86.

In terms of mechanism, CRS is the result of the extensive activation of CAR-T cells leading to 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, the patient may experience hypotension, cardiac dysfunction, circulatory failure, respiratory failure, and renal failure. Failure, failure of multiple organ systems and the possibility of death [3,4,87]. Overall, 77-93% of leukemia patients receiving CAR-T cell therapy and 37-93% of lymphoma patients receiving CAR-T cell therapy have CRS grade, while patients with relapsed/refractory B receive tisagenlecleucel 46% of patients treated with cell therapy, 13-18% of patients treated with Axicabtagene ciloleucel and tisagenlecleucel had CRS ≥ 3 [3,4]. In terms of pathophysiology, CRS is considered to be mainly mediated by IL-6, therefore, treatment relies on the use of tocilizumab and glucocorticoids for IL-6 receptor blockade [3,4,5].

Engineering CAR-T cells to improve toxicity

In order to obtain an effective therapeutic response, the CAR-T cell antigen binding domain must bind to its target epitope and reach a minimum threshold level to induce CAR-T cell activation and cytokine secretion. However, at the same time, there are some threshold levels of activation, beyond which cytokines and immune system activation will result in toxic levels. In other words, CAR-T cells must remain within their therapeutic range in order to function clinically, because too much of the therapeutic range can lead to toxicity. From an engineering point of view, CAR-T cell activation degree and activation kinetics are affected by many factors, including but not limited to the level of tumor antigen expressed on malignant cells, tumor burden, and the antigen binding domain’s effect on its target epitope. Affinity and costimulatory elements of CAR [33,92]. Therefore, it is necessary to carefully consider several components of the CAR modular structure to optimize the therapeutic effect and limit toxicity.

Change the CAR structure

One way to reduce toxicity is to change the affinity of the CAR-T cell antigen binding domain. Reducing the affinity of the antigen-binding domain is expected to lead to an increased demand for higher antigen density of tumor cells to achieve high levels of activation. Therefore, it can be expected that the reduced antigen affinity will bypass the targeting of healthy tissues with relatively small amounts of antigens. Research on this principle has shown that, compared with antigen-binding domains with low nanomolar/sub-nanomolar affinity, antigen-binding domains with micromolar affinity have higher selectivity for tumors with higher target antigen expression levels [9 ].

It is also possible to modulate the secretion of cytokines via activated CAR-T cells by modifying the hinge and transmembrane regions. For example, in CARs targeting CD19, modification of the CD8-α-derived hinge and transmembrane amino acid sequence resulted in a decrease in cytokine release levels and a decrease in CAR-T cell proliferation [93]. Optimizing the hinge and transmembrane region may be a useful way to reduce toxicity, because in phase 1 clinical trials, these modified hinge and transmembrane region CARs resulted in complete remission in 54.5% of B-cell lymphoma patients (6/11 patients) It is important that there is no CRS or ICANS event level greater than 193.

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 toxicity issues. Specifically, the 4-1BB domain leads to a lower risk of toxicity, higher T cell tolerance and a lower peak level of T cell expansion, while the CD28 costimulatory domain is associated with CAR-T cell activity. Faster onset and onset, followed by exhaustion [94]. Therefore, in tumors with high disease burden and/or high antigen density, the less toxic 4-1BB costimulatory domain may be particularly useful, while the CD28 costimulatory domain may be necessary to achieve the required T cell activation in total The low surface antigen density and/or low affinity antigen binding domain CAR94 reaches the threshold under the condition of low antigen binding domain.

CAR immunogenicity

The host immune system’s recognition of CAR constructs may be related to cytokine-related toxicity. Therefore, it may be advantageous to use human or humanized antibody fragments instead of murine CAR to reduce CAR immunogenicity [25,95]. In addition, the hinge and/or transmembrane domain can be modified to reduce the immunogenicity of the CAR, and interestingly, the persistence of CAR-T cells is improved [95,96].

Modified CAR-transduced T cells and neurotoxicity

An exciting, recently developed approach to prevent CAR-T cell cytokine toxicity is based on modifying 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, while the key large B cells Lymphoma CAR-T cell clinical trials have shown that serum biomarkers related to the development of neurotoxicity of grade 3 or higher, elevated GM-CSF are most significantly associated with neurotoxicity3. Recent preclinical studies have shown that after lenzilumabab is used to inhibit macrophage activation and monocyte activation cytokine GM-CSF, neurotoxicity and CRS are reduced, and CAR-T cell activity is increased. Mutational inactivation of GM-CSF also seems to have a similar effect in CAR-transduced T cells [98,100].

Therefore, these findings indicate that GM-CSF neutralization helps reduce neurotoxicity and reduce CRS [98]. In addition, deleting tyrosine hydroxylase in a myeloid cell-specific manner or inhibiting the enzyme with methionine will result in lower levels of catecholamines and cytokines [101]. Preclinical evidence also shows that in leukemia/lymphoma mouse models treated with CD19-targeted CARs [102], IL-1 receptor antagonists can reduce a form of neuroinflammation.

CAR “off switch”

Another potential way to improve CAR-T cytotoxicity is through the implementation of “dislocation” or suicide gene strategies. Such a strategy will promote the ability to selectively reduce engineered cells when adverse events occur through treatment with secondary inducers [103]. Several methods have been developed to utilize these concepts. For example, CAR constructs that are independently expressed or engineered to express full-length CD20 or CD20 mimotopes can promote CAR-T cell depletion by rituximab treatment [104].

However, the limitation of this method is that the start of antibody-mediated CAR-T cell depletion is relatively slow, which may limit the efficacy of this method in patients who need to be reversed immediately in severe acute cytokine-mediated toxicity. This has prompted the development of faster switches, such as the inducible cas9. In clinical trials, cas9 eliminated more than 90% of engineered T cells within 30 minutes. Other strategies rely on protease-based small molecule assisted shutdown CAR (SMASh-CAR), also known as shutdown CAR (SWIFF-CAR) [106].

The biggest limitation of suicide strategies or other similar methods is that, despite their attractiveness to ensure safety, their use abruptly stops the treatment of fast-developing diseases. This limitation strongly motivates people to develop strategies to ensure safety, while retaining suicide gene activation as a last resort. One method with exciting potential involves the use of the tyrosine kinase inhibitor dasatinib, which acts to inhibit T cell activation by inhibiting the proximal TCR signal kinase. In preclinical models, dasatinib can quickly and reversibly prevent the activation of CAR-T cells.

Administration of dasatinib as soon as possible after CAR-T cell infusion can significantly reduce the fatal CRS mortality in mice [107 ]. Therefore, this method seems to provide a temporary inhibition of CAR-T cell function, and may allow CAR-T cell therapy to be rescued after the toxicity is reduced. In the future, once the toxicity subsides, CAR-T cell therapy must be developed in the direction of first-line hematology and first-line therapy. The development of other innovative methods that can temporarily inhibit CAR-T cell function and allow CAR-T cell therapy to be rescued Malignant tumors And solid tumors.

Antigen selection is critical to CAR-T cell function. Due to the selective pressure of CAR-T cells, tumor cells can down-regulate antigens. Even with appropriate antigen targeting, extra-tumor effects on the target may occur and related toxicity may occur. In solid tumors, transporting and infiltrating CAR-T cells into the tumor is a challenge. The immunosuppressive microenvironment can exacerbate this obstacle. Effective treatment also has 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 therapies in the future.

(source:internet, reference only)


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