CAR T-Cells in Acute Lymphoblastic Leukemia: Current Status and Future Prospects

The currently available treatment for acute lymphoblastic leukemia (ALL) is mainly dependent on the combination of chemotherapy, steroids, and allogeneic stem cell transplantation. However, refractoriness and relapse (R/R) after initial complete remission may reach up to 20% in pediatrics. This percentage may even reach 60% in adults. To overcome R/R, a new therapeutic approach was developed using what is called chimeric antigen receptor-modified (CAR) T-cell therapy. The Food and Drug Administration (FDA) in the United States has so far approved four CAR T-cells for the treatment of ALL. Using this new therapeutic strategy has shown a remarkable success in treating R/R ALL. However, the use of CAR T-cells is expensive, has many imitations, and is associated with some adverse effects. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are two common examples of these adverse effects. Moreover, R/R to CAR T-cell therapy can take place during treatment. Continuous development of this therapeutic strategy is ongoing to overcome these limitations and adverse effects. The present article overviews the use of CAR T-cell in the treatment of ALL, summarizing the results of relevant clinical trials and discussing future prospects intended to improve the efficacy of this therapeutic strategy and overcome its limitations.


Introduction
Acute lymphoblastic leukemia (ALL) is a rare cancer in which there is an abnormal clonal proliferation of early lymphoid stem cells along with the depletion of normal bone marrow hematopoietic cells [1].The two major types of ALL are B and T lymphoblastic leukemia.While precursor B-cell ALL (B-ALL) represents the majority of ALL cases, T-cell neoplasm tends to behave more aggressively [2].In 2021, around 6000 cases of ALL were reported in the United States.Children and young adults' affection represent around 80% of these cases [3].Combination of chemotherapy, steroids, and allogeneic stem cell transplantation (Allo-SCT) is the mainstay for treating this type of leukemia [4].While such therapeutic protocols were able to increase long-term survival rates to around 90% in pediatric ALL [5], refractoriness and relapse (R/R) after initial complete remission (CR) were reported in up to 20% of the cases [2,6].In comparison to pediatric ALL, the figures are worse in adult leukemia.It was reported that the percentage of adults with ALL who achieved long-term disease-free survival was only 40% [7].After relapse, the reported overall survival was only about 7% [8].Such facts urge the need for newer therapeutic approaches to overcome R/R.One of these approaches that has been developed over the past few years is the use of chimeric antigen receptor-modified (CAR) T-cell therapy.

What Is CAR T-Cell Therapy?
CAR T-cells were developed and proposed as a potential therapeutic strategy by Gross and colleagues more than 3 decades ago [9].The use of these cells entails the genetic modification of T-cells in order to enhance their ability to express cell surface synthetic receptors which are capable of interacting with specific antigens expressed on the cancer cells [10].CARs are fusion proteins.The primary structure of a CAR consists of an extracellular tumor antigen-binding domain called the ectodomain, a transmembrane domain, and an endodomain which is an intracellular signaling domain responsible for T-cell activation [10,11].The striking difference between CAR T-cells and unmodified T-cells is that CAR T-cells do not require human major histocompatibility complex (MHC) molecules to recognize antigens on the surface of tumor cells.CAR T-cells have the ability to directly attack cancer once these cells bind to the specified antigen located on the surface of tumor cells [10].
Currently, there are four generations of CAR T-cells.An additional fifth generation is in the pipeline.The design of the first generation of CAR T-cells was simple.The endodomain responsible for the activation of T-cells was formed of CD3ζ chain only [12].Such a simple structure enabled only the activation of T-cells without being capable of supporting their cellular expansion.Thus, the therapeutic efficacy of this early generation was limited [9].In fact, three signals are required to optimize T-cell activation and proliferation.Using CD3ζ chains in the first generation only ensure signal 1, which is known as T-cell receptor engagement.The other two signals are co-stimulation, which is called signal 2, and cytokine engagement, which is known as signal 3 [13].The lack of co-stimulation (signal 2) was then addressed in the second and third generations of CAR T-cells.The addition of one or two co-stimulatory signaling domains to the endodomain of the second and third generation, respectively, fulfilled the function of signal 2 [9,14].The use of the signaling domains of CD28, CD134, or CD137 incorporated to CD3ζ chains has significantly enhanced the activity, expansion, and survival of these modified T-cells and even stimulated interleukin (IL)-2 production [15][16][17].The fourth generation is much more sophisticated.Modification of the structure to allow remote-controlling of CAR T-cells activity and limiting their toxicity is one example [18].Modifications to overcome target antigen loss or down-regulation, which results in tumor escape [19,20], or to increase the CAR T-cell specificity by requiring the presence of two antigens on the same cell to allow T-cell activation [21,22], have also been reported.CAR T-cells may also be modified to enhance their ability to produce inducible cytokines, as IL-12 or interferon (IFN)-γ, which can modulate the tumor microenvironment and enhance CAR T-cells function [23,24].These cells are known as CAR T-cells redirected for universal cytokine killings (TRUCKs), and they have demonstrated a significant ability to overcome target antigen loss/antigen-negative relapse on the surface of cancer cells [23].The fifth generation is still in the exploratory stage.This generation is designed to cover cytokine engagement (signal 3).CAR T-cells belonging to this generation have the ability of inducing cytokine signaling upon antigen stimulation.CAR T-cells of this generation encode a truncated cytoplasmic domain from the IL-2 receptor β-chain with a transcription factor.These cells showed antigen-dependent activation and superior antitumor effects with significantly minimized toxicities.It is also expected that these cells would have a better outcome against antigen-positive relapse [25,26].Of note, none of the third, fourth, or fifth generations of these modified T-cells have been approved yet for clinical use.

Manufacturing of CAR T-Cells
The manufacturing of CAR T-cells starts first with the isolation of peripheral blood mononuclear cells from the patient (autologous) or a healthy donor (allogeneic) using leukapheresis.The next step entails the use of specific antibodies to separate the target T-cell subsets, which are then activated by beads coated with anti-CD3/anti-CD28 monoclonal antibodies, anti-CD3 antibodies, or artificial antigen-presenting cells (APCs).The activation process may be enhanced by cytokines, such as IL-2.The following step is the process of gene transfer of the CAR construct to the primary T-cells.Owing to the lower risk of mutagenesis, lentiviral vectors are preferred for gene transfer over retroviral vectors [27,28].Non-viral approaches for gene transfer have also been used in this step.The designated CAR T-cells are subjected to expansion and phenotyping, and then they will be cryopreserved for subsequent infusion [28][29][30].

CAR T-Cells for ALL
There are several antigens on ALL cells that have been targeted in different experimental and clinical trials on CAR T-cell therapy.CD19 has received most of the attention in B-ALL.It is constantly expressed throughout B-cell development.In addition, CD19 is not expressed on hematopoietic stem cells.This will reduce the chance of developing aplastic anemia while using this therapeutic strategy [31].Still, targeting CD19 could possibly result in B-cell aplasia with a consequent long-lasting defect in humoral immunity and recurrent respiratory tract infections [32,33].At the moment, all the currently FDA-approved CAR T-cells for treating ALL are targeting CD19 (Table 1).• N.B.None of these therapeutics could be used to treat patients with primary central nervous system lymphoma.

•
DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; R/R: relapsed/refractory.CD20 is another target antigen under investigation as it is specific for B-cell lymphoid malignancies [38,39].Similarly, the receptor for thymic stromal lymphopoietin (TSLPR) was also considered as a promising target for CAR T-cell therapy in selected cases of B-ALL [40,41].For T-cell ALL, CD7-based CAR T-cells are being investigated in xenograft models, and promising results have encouraged an ongoing clinical trial [42,43].Similarly, CD38-based CAR T-cells have been designed to target HTLV-1 + T-cell leukemia [44][45][46].Other than targeting only one antigen on the surface of ALL cells, the use of multiple targets, as in the fourth generation of CAR T-cells, is still under investigation in order to reduce on-target, off-tumor toxicities [21,22] and prevent tumor antigen-loss relapses [19,20].

Limitations and Toxicities of CAR T-Cell Therapy
The first barrier to accessing CAR T-cell therapy is probably the cost.In the United States, it was estimated that one infusion of CAR T-cells can cost between USD 373,000 and 475,000 [47].Patient care and disease monitoring can increase the number by USD 79,466 to 85,267 [48].Moreover, the management of CAR T-cell toxicities and post-CAR T-cell therapy events can increase the total cost to exceed USD 1 million for some patients.As an average, the cost for this new therapeutic strategy can range from USD 500,000 to 600,000 [49,50].Several technical and regulatory issues along with institutional infrastructure readiness might also be limiting factors for the use of CAR T-cell therapy [51].Away from the high cost barrier, the two major limitations for CAR T-cell therapy are failure of treatment and toxicity.

Failure of CAR T-Cell Therapy
Treatment failure is a major limiting factor for the use of CAR T-cell therapy.The incidence of relapse among B-ALL patients who received treatment with CAR T-cells showed a wide variation in different studies and ranged between 22% and 75% [52].Considering that only anti-CD19 CAR T-cells are the currently FDA-approved CAR Tcell therapy for B-ALL, we can classify relapse to either CD19-positive or CD19-negative relapses.Antigen-positive relapse is more common and is mainly related to T-cell potency and its in vivo expansion ability.Finding that the rate of B-ALL relapse is less in children compared to adult patients suggested that the age of T-cell might be a major determinant for antigen-positive relapse [53].The use of co-stimulation/signaling domains and other techniques to enhance the activity, stimulate the expansion, and extend the survival of CAR T-cells has been suggested, and some have shown a promising ability to reduce antigen-positive relapse [17,26,[54][55][56].On the other hand, antigen-negative relapse or tumor antigen escape is more relevant to tumor cells.Generally, loss of CD19 antigen is responsible for about 10-20% of relapses in patients with B-ALL [57].However, some clinical trials reported higher percentages [58].Antigen-negative subclones might pre-exist even before starting CAR T-cell therapy and cause R/R [59].Several mechanisms have been proposed to explain the antigen-negative relapse.While gene mutation, pre-treatment with blinatumomab, and RNA splice variants [60][61][62] can explain most of the cases of CD19-negative relapse, down-regulation of the target antigen can take place with other CAR T-cell therapies of B-ALL as anti-CD22 CAR T-cell therapy [63].That is why the design of many of the fourth generation CAR T-cells is addressing the relapse due to antigen loss.Designing CAR T-cells with dual CD19/CD123 targeting was reported to prevent antigen-negative relapses [20].Still, the genetic instability and heterogeneity of antigen expression on the leukemic cell surface, especially in the case of high tumor burden, can lead to another relapse even after dual-target CAR T-cell therapy [64][65][66].Other B-cell lineage markers, as CD20 and CD22, have also been targeted, and epigenetic modifying agents have also been tested to overcome the immune evasion of tumor cells during therapy with CAR T-cells [67][68][69].

Cytokine Release Syndrome
The toxicity of CAR T-cell therapy can take many forms.One of the most important and most common toxicities related to the use of this relatively new therapeutic strategy is the cytokine release syndrome (CRS) [70].In children with B-ALL, it was reported that the incidence of CRS can reach up to 77% [58].In adults, figures are much higher and ranged from 85 to 93% depending on the study [53,71].In CRS, the activation of T-cells will lead to a significant elevation of the inflammatory cytokines (e.g., IL-6, IL-10, IFNγ, and GM-CSF) [72][73][74].Patients are usually presented with fever, headache, myalgia, hypotension, and hypoxia.The condition may be complicated with cardio-respiratory dysfunction or even failure.The burden of the disease and the used dose of CAR T-cells are major determinant of the severity of the CRS [72,73].The management of CRS is largely dependent on the disease grading.Anti-cytokine therapy is the mainstay for treating CRS together with proper supportive treatment for the associated hypotension and/or hypoxia.IL-6 receptor blockade via tocilizumab with or without systemic corticosteroids is usually needed in grade 2 CRS and onward.Siltuximab, an anti-IL-6 monoclonal antibody, and anakinra, an IL-1 receptor antagonist, have been suggested as alternatives to tocilizumab.Most patients respond well after therapy, yet CRS symptoms may persist in very rare cases [75][76][77].

Immune Effector Cell-Associated Neurotoxicity Syndrome
Another common toxicity associated with the use of CAR T-cells is known as immune effector cell-associated neurotoxicity syndrome (ICANS).Patients may show a wide range of presentation varying from confusion and delirium to motor dysfunction, expressive aphasia, tremor, and ataxia.Rare cases presented with myoclonic seizure and even fatal brain edema [72][73][74].The incidences of these presentations also vary widely between studies and are largely affected by the used grading scale [78,79].The precise mechanism of these neurologic adverse effects is unknown, but it is likely to be related to the elevated levels of different inflammatory cytokines.That is why such syndrome usually follows CRS [80].As CD19 is expressed on the left inferior frontal gyrus, it is possible that ICANS is part of the on-target/off-tumor effect [81].However, reporting similar neurotoxicity with CAR T-cells targeting other antigens may suggest that ICANS is not antigen-specific [82,83].The disease is self-limited and may resolve even without any intervention [84].Unlike CRS, tocilizumab has no role in ICANS and may even worsen the neurological manifestations.If needed, treatment with corticosteroids in addition to standard care treatment for neurological presentation, such as seizures, can reverse the disease in the majority of patients.Still, few mortalities have been reported with ICANS, especially when the cases were associated with cerebral edema [75][76][77].Of note, both CRS and ICANS are more likely to take place when anti-CD19 CAR T-cells were used for B-ALL in comparison to their usage in other tumors [85].

Infection and Hypogammaglobulinemia
Infection is another complication to the use of CAR T-cells in leukemia.The reported incidence of infection with CAR T-cell therapy is around 43% [58].Early infection is usually bacterial and is probably due to the immunosuppressive effect of lymphodepleting chemotherapy which is given before CAR T-cell infusion.Late infection is more relevant to CAR T-cell therapy and is more likely to be viral.This is attributed to CAR T-cell therapy itself in a phenomenon called "on-target/off-tumor recognition".As the target antigen for CAR T-cell is shared by normal cells, it is possible that non-pathogenic immune cells expressing the target antigen (CD19) will also be affected by the used CAR T-cells.The delayed recovery of these cells will result in significant hypogammaglobulinemia [86][87][88].The condition could be fatal and requires IgG replacement to protect against infectious complications [86,88].

Other Toxicities of CAR T-Cell Therapy
Another immunological dysfunction that might take place after CAR T-cell therapy is called hemophagocytic histiocytosis (carHLH) [75,89].The mechanism of carHLH is unknown but involves the production of pro-inflammatory cytokines, lymphohistiocytic tissue infiltration, and immune-induced multiorgan damage and dysfunction.Until the proper patient identification and management protocols are standardized, the current management of carHLH is identical to severe CRS cases [77].Other toxicities that have been reported with CAR T-cell therapy include cerebral edema, cardiotoxicity, allergic reaction, and tumor lysis syndrome [77].

Clinical Trials Using CAR T-Cells in ALL
A long list of clinical trials which are investigating the efficacy and safety of CAR Tcells in the treatment of ALL are ongoing/recruiting, completed, or even withdrawn [85,90].We have listed the results of some of these clinical trials in Table 2. Other than clinical trials, several studies have been published to evaluate collective data obtained from related previous clinical trials.One example is the study of Pasquini et al. [91], who collected the safety and efficacy data of a CD19-targeting CAR T-cell therapy: tisagenlecleucel (tisacel/Kymriah).The data of 410 patients have been evaluated.There were 255 ALL patients, while the remaining 155 were non-Hodgkin lymphoma patients.The initial cure rate among ALL patients was 85.5%.Overall survival at 12 months was 77.2%.Adverse effects as grade-3 CRS accounted for 16.1% of the patients, while grade-3 ICANS was about 9.0%.These figures are much lower than those reported in some of the key studies, probably due to using different grading scales for the assessment of these adverse effects.In a parallel context, we have found one meta-analysis of several clinical trials that reported the safety and efficacy data of anti-CD19 CAR T-cells.Pooled data of 953 R/R B-ALL patients were presented, and the pooled CR was about 80%.Of note, CR was significantly improved when autologous CAR T-cells were used in comparison to allogeneic CAR T-cells.However, using autologous CAR T-cells was more likely to cause ICANS.In addition, the percentages of patients having CRS or ICANS were not significantly affected by the use of different anti-CD19 CAR T-cell constructs [92].
Of note, most of the clinical trials listed in Table 2 were using CD19-targeting CAR T-cells; few trials tested other targets.Investigating other targets would be helpful in case of relapse after using CD19-targeting CAR T-cells.In the study of Pan and his colleagues, they used CD22-targeting CAR T-cells in patients with B-ALL who failed to respond to CD19 CAR T-cell therapy.Among the 34 enrolled patients, 24 patients achieved CR or CR with incomplete count recovery after 30 days.Seven CR patients did not receive any additional treatment, and three of these patients remained in remission after 14 months.Transplantation was conducted in 11 CR patients, and 8 of them remained in remission at 4.6 to 13.3 months.Adverse effects were mild in most of the patients, and CD22 antigen mutation or loss was not reported even in patients with reported relapse [93].Using dualtarget CAR T-cells was also subjected to clinical testing to overcome relapses that might take place after the use of CD19-targeting CAR T-cell therapy.In one study, bispecific CAR T-cells targeting both CD19 and CD22 were used in patients with R/R B-ALL.All the six enrolled patients achieved CR.No neurotoxicity was reported, but one case of relapse took place 5 months later where the blast cells of the patients lost CD19 expression and exhibited diminished CD22 site density [94].Subsequent studies enrolled more patients to enhance the reliability of the results [95,96].A recently published meta-analysis evaluated the efficacy and safety of CD22 or CD19/CD22 CAR T-cells in the treatment of ALL and NHL.In ALL, CD19/CD22 CAR T-cells had a CR rate of about 90%.The incidences of total and severe CRS were 87% and 6%, respectively.The incidences of ICANS and severe ICANS were 16% and 3%, respectively [97].50% hypotension 42% anemia 33% grade 3 CRS 21%, 25%, 27%, and 11% neurological events depending on the dose [114] ALL: acute lymphoblastic leukemia; CR: complete remission; Cri: complete remission with incomplete count recovery; CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxicity syndrome; NHL: non-Hodgkin lymphoma; Nr: not reported; OS: overall survival; R/R: relapsed/refractory.N.B.Whenever more than one article described the same clinical trial, we used to include the results of the latest one and point to relevant articles where preliminary data were described.

Future Prospects for CAR T-Cell Therapy in the Treatment of ALL
Although three decades have now lapsed since CAR T-cell therapy was first introduced as a new therapeutic modality, efforts to improve this therapeutic approach are still ongoing to overcome its limitations.One of these limitations is the time needed to manufacture personalized T-cells.Such a process can take up to 3 weeks and delay treatment [115,116].This has led to the development of what is known as "off-the-shelf" CAR T-cells.
Off-the-shelf CAR T-cells are newly developed universal allogeneic CAR T-cells where T-cells are collected from healthy donors.This ensures that a good number of healthy T-cells are retrieved and, unlike autologous CAR T-cells, are not affected by chemotherapy or cancer cells [117,118].It was suggested that CAR T-cells performance could be greatly affected by the quality and quantity of retrieved T-cells [53,119].Thus, we might expect that the performance of such produced allogeneic CAR T-cells would be improved in comparison to autologous cells.Off-the-shelf CAR T-cells, in addition to ensuring goodquality cells, will also facilitate the large-scale production of T-cells, which saves both money and time.It allows the patient to have immediate access to the treatment.There is no need to wait for the 3-week interval which is usually required to manufacture personalized autologous CAR T-cells [115,118].The term (FasTCAR) is gaining popularity and is used to indicate the next-day manufacturing of CAR T-cells using novel manufacturing platforms.The results of clinical trials on patients with relapsed ALL indicated the superior expansion capacity of these allogeneic CAR T-cells and comparable efficiency with manageable toxicity profile [120].However, one meta-analysis reported that while neurotoxicity was reduced with allogeneic CAR T-cells, CR rates were higher when autologous CAR T-cells were used in comparison to allogeneic CAR T-cells [92].In addition, there is another major drawback of off-the-shelf CAR T-cells.Using T-cells from a donor with MHC-mismatching can be complicated with graft rejection and the serious and life-threatening complication, graft-versus-host disease (GVHD).
In graft rejection, the remaining functioning host immune cells will attack and eliminate the transferred CAR T-cells and compromise their antitumor efficacy [121].Rejection of allogenic CAR T-cells can be prevented in several ways.One way is to disrupt certain components of the MHC Class I of the allogeneic CAR T-cell.This might prevent the allogenic rejection by the host cells [122][123][124].Another approach is to delete CD52 on the surface of CAR T-cells.At the same time, the anti-CD52 monoclonal antibody, alemtuzumab, is used to deplete the host T-cells before CAR T-cell infusion.Thus, the infused T-cells evaded the rejection and simultaneously will not be targeted by alemtuzumab.Such a strategy was successful but was hindered by the increased risk of cytopenia and severe viral infections [125,126].Another way to prevent the rejection of transferred allogenic CAR T-cells is to engineer a chimeric receptor, called the alloimmune defense receptor (ADR), that enables used CAR T-cells to recognize and eliminate alloreactive lymphocytes while saving the host resting lymphocytes in peripheral blood.It was found that the co-expression of ADR and CAR on the engineered T-cells conveyed resistance to allogenic rejection, while the anti-tumor activity was not compromised [121,127].
On the other side, different strategies have been tested to bypass GVHD.Using third-party allogeneic virus-specific T-cells is one example that has been suggested based on the encouraging data when these cells were used to treat post-transplant viral infections [128][129][130][131]. Another method to overcome GVHD is the use of T-cells that are genetically modified.The genetic modification of T-cells entails the removal of endogenous molecules, as αβ T-cell receptors, in order to make the produced T-cells unable to mount the usual alloreactive immune response against the recipient's normal tissue.Genetic modification could be achieved using different gene editing techniques, such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins or transcription activator-like effector nucleases (TALENS) [122][123][124]132].Such a strategy showed some success in small clinical studies [125,126,133].Instead of removing the αβ T-cell receptors, using a minor T-cell subset called γδ T-cells could be also promising to overcome GVHD.These cells represent only 5-10% of the total population of T-cells but have a striking feature in the way that the expression of γδ T-cell receptors on the surface of these cells is MHC-independent.This feature made this subtype of T-cells a suitable candidate for off-the-shelf CAR T-cell production with minimal risk of GVHD [134].
Another strategy to circumvent GVHD, other limitations, and the toxicities of CAR T-cells is to replace T-cells with an alternative effector cell.In this context, Natural killer (NK) cells and macrophages might be perfect targets for producing off-the-shelf CARs.These cells, unlike T-cells, are components of innate immunity, and their action is independent of MHC.Thus, it is unlikely that they may cause GVHD.Moreover, antigen escape is also unlikely to take place with NK-based CARs.These cells have the ability to recognize cancer cells independent of MHC and will keep their potency even if MHC molecules are down-regulated [135].Indeed, CAR-NKs have shown promising results with minimal toxicities in several models of hematological and solid tumors, as well as one clinical trial [136].Several other clinical studies have been registered, though the results are yet to be announced [137].For macrophages, they have several advantages that can make them suitable for CARs.These cells, by selective phagocytosis, destroy cancer cells and then activate adaptive immunity by presenting the antigens to T-cells.Macrophages, by enhancing the secretion of pro-inflammatory cytokines, can also potentiate the cytotoxicity of T-cells against tumors [138,139].In addition, these cells can easily infiltrate the tumor microenvironment and secrete cytokines that modulate the suppressive microenvironment generated by tumor cells [139].Macrophages-based CARs showed some success in preclinical studies, and clinical trials are being conducted [138,140].
Future prospects in CAR T-cell therapy also entail the search for newer targets.As we previously discussed, all the currently available treatments for B-ALL are based on targeting CD19.Thus, tumor relapse is a common limitation for all currently available CAR T-cell therapy as a result of the down-regulation of CD19 on the surface of cancer cells [57,58].For this reason, many of the recent studies actually focused on the search for new targets, such as CD20, CD22, and BCMA [67,69,82,141].The success of these newer targets in comparison to targeting CD19 remains questionable.It was suggested that CD19 expression is more stable than other targets.Such stability could allow for obtaining better antitumor activity in comparison to other antigens [142].However, one clinical trial was conducted on B-ALL patients using a relatively low dosage of CD20 CAR T-cell therapy.The study reported that targeting CD20 resulted in the induction of a high remission rate and low toxicities in patients who relapsed after CD19 CAR T-cell therapy [66].Another new method to avoid CD19-negative relapses is to use dual-target CARs.The dual-target CARs strategy entails simultaneous targeting CD19 and another antigen.For example, the presence of either CD19 "OR" CD20 on target cells can fully induce the immune response of anti-CD20-CD19 bispecific CAR T-cells in preclinical settings [143].Bispecific CAR T-cells could also be designed to work upon the simultaneous presence of both antigens, for example, CD19 "AND" CD22 [94,144,145].The dual targeting of CD19 and CD123 or B-cell activating factor receptor (BAFF-R) has also been tested [146,147].CAR T-cells which are targeting three antigens, CD19/CD20/CD22, are an even more sophisticated strategy being tested for the treatment of ALL.Initial results showed that these trivalent CAR T-cells were more effective in killing ALL cells, including CD19-negative ones [148].In a parallel context and rather than using dual/trivalent targeting CAR T-cells, Yan et al. used a sequential infusion of two different CAR T-cells, anti-CD19 and anti-CD22, in patients with relapsed ALL after hematopoietic stem cell transplantation (HSCT).This trial has shown that the use of such a cocktail was safe and effective for relapsed cases of ALL [149].

CAR T-Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL)
Another indication for the use of CAR T-cell therapy is to target T-ALL.As we mentioned earlier, T-ALL is a rare but aggressive tumor with a very poor prognosis [2,150].One major limitation that hindered the progress of developing CAR T-cells against T-ALL is that the T effector cells, which is used as a treatment, share the expression of almost all the antigens as malignant T-cells [151,152].This will result in self-killing or what is known as the fratricide of CAR T-cells.Fratricide can reduce the proliferation of CAR T-cells in vitro and compromise their action after being infused.Several strategies have been tested to overcome this obstacle.As we discussed earlier, replacing T-cells with NK cells is one strategy.Several preclinical studies used CAR-modified NK cells to treat T-ALL, and the results were indeed encouraging to conduct clinical trials using these cells [153][154][155].Another strategy to avoid fratricide is to find a tumor-specific antigen with a restricted expression to malignant T-cells only.As reviewed by Ren and his colleagues [156], several biomarkers have been identified and can be targeted by CAR T-cells depending on the limited expression of these biomarkers on normal T-cells.CD7 or CD38-based CAR T-cells are relevant examples [42][43][44][45][46].While some studies showed a successful generation of anti-CD147-CAR T-cells that conveyed protection against T-ALL progression in a xenograft model [157], others have been tested clinically.For example, nanobody-derived CD7-CAR T-cells showed a remarkable ability to induce remission in one case of an 11-year-old male patient with R/R early T-ALL [158].

Conclusions
CAR T-cell therapy appears to be a promising new therapy for ALL, especially cases with R/R, as evident in several experimental and clinical settings.However, as with any therapeutic modality, using CAR T-cells is associated with several limitations and toxicities.In addition, the process of manufacturing these personalized T-cells is expensive and time-consuming.Continuous development of this therapeutic strategy is ongoing to overcome these limitations and adverse effects, reduce cost, and ensure the availability of this therapeutic approach in the proper time.

Table 2 .
Selected clinical trials that investigated the safety and efficacy of using CAR T-cells in the treatment of acute lymphoblastic leukemia (ALL).