Recently, studies have shown that IL-1 and IL-6 released by monocytes and macrophages are the main reasons for CAR-T-associated CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) (66, 67). In detail, monocytes play a more important role than macrophages in CRS-progressing IL-1 and IL-6 secretion (67). Preclinical data indicate that monocyte ablation or IL-6 receptor blockade using tocilizumab can prevent CRS but not lethal neurotoxicity (67). This is because IL-6 receptor blocking antibodies broadly used as the standard procedure for the clinical management of severe CRS cannot penetrate the BBB which results in their inability to induce complete CAR-T-related encephalopathy syndrome (CRES) remission (67). Furthermore, CRS and CRES are the major safety-related issues of CAR-T therapy which are caused by a rapid increase in the level of multiple cytokines, mainly IL-6, secreted by the activated infused CAR-Ts and mononuclear cells including dendritic cells and macrophages (68). In detail, target cell-induced IL-6 expression and production by CAR-Ts trigger factors that enhance monocyte production and release of IL-6 and other proinflammatory cytokines contributing to CRS and CRES progression (69). Therefore, some researchers have investigated a very novel strategy to address these limitations (69–71). It has been discovered that suppressing the IL-6 gene expression in CAR-Ts significantly decreases the IL-6 release from monocytes and it also reduces the possibility of severe CRS and CRES without reducing the antitumor efficacy of CAR-Ts in vitro and in vivo (69, 70). Therefore, one research group has generated a CAR construct with IL-6 shRNA (70). The shRNA-IL6-modified CD19-redirected CAR-Ts (ssCAR-T-19) were delivered to a B-ALL patient with skin and testicle extramedullary relapse enrolled in a clinical trial (NCT03919240) (70). The patient was successfully treated by ssCAR-T-19s and this study proposed that these CAR-Ts might be therapeutically useful in efficiently eradicating infiltrating leukemia cells in the skin and testicle with a mild level of toxicity (however, broader patient populations and more careful clinical investigations are required for drawing substantiated conclusions) (70). Furthermore, ssCAR-T-19s have also been investigated in a recent clinical trial (NCT03064269) involving three patients with relapsed central nervous system (CNS) B-ALL (71). The results of this study demonstrated that ssCAR-T-19s can migrate into the CNS and reduce brain leukemic infiltration and significantly eradicate leukemic blasts in the cerebrospinal fluid (CSF) with mildly elevated cytokine levels and only grade 1 CRS (71). Therefore, this method could be proposed as an approach for reducing the risk of CRES in CAR-T therapy of patients with CNS B-ALL (71). However, in the case of severe CRS occurrence, IL-6 receptor blockade or IL-6 expression inhibition strategies are known to be inadequate approaches (16, 72). Therefore, it is necessary to use high-dose corticosteroid drugs to efficiently control the progression of life-threatening CRS (16, 72). Moreover, recent studies have shown that corticosteroids do not influence the efficacy and kinetics of CAR-Ts in the treatment of B-ALL (73). Additionally, TO-207 is an investigational drug that can inhibit the abnormal activation of macrophages and monocytes with no negative effects on the activity of T cells (74–77). In vitro studies have demonstrated that TO-207 acts as a multi-cytokine inhibitor by suppressing monocyte-mediated secretion of IL-6 and other inflammatory cytokines (including IL-1β, monocyte chemoattractant protein 1 (MCP-1), IL-8, IL-18, and GM-CSF) without deteriorating CAR-T functionality (74, 78). Furthermore, IL-1 receptor blockade using the IL-1 receptor antagonist immunosuppressive drug anakinra has been an effective approach resulting in encouraging preclinical outcomes by preventing both lethal neurotoxicity and CRS (67). The same study has also claimed that anakinra and tocilizumab exhibit the same level of effectiveness in IL-6 signaling blockade and lethal CRS prevention in preclinical models (67). Moreover, CAR-Ts particularly engineered to secrete IL-1 receptor antagonists have also been investigated in preclinical studies (66). The results have indicated that IL-1 receptor antagonist-secreting CAR-Ts can prevent or mitigate CRS as well as neurotoxicity in a very efficient manner (66). As discussed, IL-1 blockade strategies can be efficiently utilized to prevent life-threatening CAR-T-related toxicities including CRS and neurotoxicity (66). However, it is important to mention that conducting clinical investigations with a high number of patients is a vital step in further validating the preclinical and clinical outcomes discussed in this section.

Neurotoxicity and CRS development after CAR-T therapy are directly related to in vivo T-cell expansion and subsequent secretion of T-cell effector cytokines such as IL-6, IFN-γ, GM-CSF, and MCP-1 suggesting that even monocytes and macrophages may have contributions in the emergence of such adverse events (15, 79–82). Therefore, the neutralization of such cytokines may be a potential strategy for the management of these CAR-T-related toxicities (79, 83, 84). GM-CSF neutralization reduces the secretion level of CRS-mediating cytokines such as IL-6, IL-8, and MCP-1 (79, 83, 84). Therefore, reductions in the level of these immune cell trafficking mediators can decrease the number of CNS-infiltrating immune cells which results in a reduced level of neuroinflammation (NI) and CRS (79, 83, 84). Sterner et al. have evaluated the effects of GM-CSF neutralization on the functionality of CD19-redirected CAR-Ts using lenzilumab and they have demonstrated that lenzilumab does not inhibit the function of the CAR-Ts (79). Moreover, they have reported enhanced CD19-redirected CAR-T proliferation and durable control of leukemic disease in patient-derived xenograft (PDX) models after lenzilumab-mediated neutralization of GM-CSF (79). Additionally, these researchers expanded their experimental zone by generating CRISPR-Cas9-mediated GM-CSF secretion-deficient (GM-CSF^k/o) CD19-redirected CAR-Ts (79). These GM-CSF^k/o CAR-Ts were capable of maintaining their normal functions, exhibiting enhanced in vivo tumoricidal activity, and improving OS as compared with conventional CD19-redirected CAR-Ts (79). Other studies have used other genome-editing strategies such as the transcription activator-like effector nuclease (TALEN) for the same aim (85). They have demonstrated that their GM-CSF^k/o CAR-Ts can mediate a substantially reduced secretion level of GM-CSF leading to a disrupted or decrease macrophage-dependent secretion of CRS-mediating factors such as MCP-1, IL-6, and IL-8 (85). Moreover, CAR-Ts can be engineered to be deficient in GM-CSF expression and produce anti-GM-CSF mAbs. The mentioned findings demonstrate that GM-CSF inhibition might be considered as a potential option for the abrogation of CRS and NI and enhancement of CAR-T function (79). However, it is safe to conclude that in-depth clinical assessments might help researchers understand the suitability of such strategies in clinical settings.

Myeloid-derived catecholamines are known as crucial mediators of CRS (86–88). Macrophages secrete and respond to catecholamines via their adrenergic receptors upon encountering inflammatory stimuli (86–88). This mechanism results in an increase in the level of cytokine production and secretion (87–89). Therefore, high levels of circulating catecholamines can lead to excessive inflammatory responses (87–89). It has been found that disrupting the key enzymes involved in the synthesis of catecholamines can reduce its circulating level and prevent CRS occurrence (90, 91). For example, myeloid-specific deletion of tyrosine hydroxylase (TH) using metyrosine (MTR) can reduce the excessive level of this key CRS mediator without impairing CAR-T antitumor activity (91). Additionally, atrial natriuretic peptide (ANP) can also significantly reduce the circulating level of catecholamines without any interference with CAR-T-mediated therapeutic responses (90).

As key CRS modulators, inflammatory cytokines including ILs, IFNs, and several growth factors crucially require the JAK tyrosine kinase family for their downstream signaling pathway (92–99). Therefore, the inhibition of the JAK family components can lead to a reduced CRS-related cytokine level (92–99). Itacitinib (INCB039110) is a JAK1 inhibitor that is being evaluated in clinical trials for the treatment of GvHD (NCT02614612) (99) and in preclinical investigations for the treatment of inflammatory and autoimmune diseases (97, 98). Itacitinib has also been evaluated in combination with other drugs for the treatment of various B-cell malignancies in clinical trials (NCT02018861 and NCT01905813) (95, 96). Recently, studies have evaluated the effects of prophylactic itacitinib in the prevention of CAR-T therapy-mediated CRS, and they have found that itacitinib does not impair CAR-T functionality both in vitro and in vivo and it significantly reduces CRS-associated cytokine levels in a dose-dependent fashion (94). Such data suggest the potential of itacitinib as prophylactic therapy for the prevention of CAR-T-related CRS as other studies investigated its various safety aspects (93). Additionally, ruxolitinib is also another JAK inhibitor that has recently been used as adjuvant therapy in a case report study involving a patient with B-ALL (92). Ruxolitinib has been successful in controlling corticosteroid-resistant CRS without interfering with CAR-T functionality (92). Such studies highlight the role of JAK inhibitors in the prevention and control of CRS and pave the way for more in-depth preclinical and clinical investigations.

IT chemotherapy has been used for controlling refractory CAR-T-associated ICANS that is non-responsive to steroids (100, 101). Shah et al. have administered IT chemotherapy to two CAR-T-receiving patients and demonstrated that it can mediate rapid ICANS resolution without any long-term complications (100). This method can also accelerate the recovery process and reduce the complications of systemic long-term corticosteroid administration (100). Moreover, Yucebay et al. have also reported similar results from a study where IT chemotherapy resulted in the resolution of high-grade neurotoxicity (grade 3-4) of CD19-redirected CAR-T-receiving patients while corticosteroid therapy was inefficient (101). However, both of these studies suggest that this method requires detailed evaluations in future clinical trials (100, 101).

The occurrence of CRS and neurotoxicity seem to have a direct relationship with the disease burden of patients receiving CAR-T therapy (14, 16, 17). Pretreating patients with antibody-based immunotherapy such as blinatumomab or inotuzumab ozogamycin (IO) before the administration of CAR-Ts has been known to potentially reduce disease bulk, thus minimizing the incidence and severity of the mentioned toxicities (102). Blinatumomab is a T-cell-redirecting bispecific antibody (TRBA) that can potentially mediate the elimination of leukemic cells through the engagement of endogenous T cells by simultaneously binding to CD19 on the surface of B-lineage cells and CD3 on cytotoxic T cells (103). Moreover, IO is an antibody-drug conjugate consisting of a humanized mAb against CD22 conjugated to the cytotoxic antibiotic agent ozogamycin (104). These therapeutic antibodies have shown considerably improved OS rates in comparison with standard therapy (103, 104). Additionally, one study including five adult patients with B-ALL (NCT02772198) has reported that treatment of R/R B-ALL patients with blinatumomab and/or IO before the beginning of CD19-redirected CAR-T therapy can result in promising response rates (with two out of five patients (40%) achieving minimal residual disease (MRD)-negative CR and two other patients (40%) achieving MRD-positive CR) (102). However, further investigations involving broader patient populations are required for a better understanding of the action mechanisms of antibody-based treatments before CAR-T therapy and their effects on the final clinical outcomes.

Recently, it has been shown that TPE ( Figure 2A ) in combination with glucocorticoid therapy can result in the gradual resolution of the CRS-related symptoms of CAR-T therapy recipients (105, 106). These findings suggest that this strategy can be a feasible procedure, at least in patients with severe CRS (grade ≥ 3), even though TPE is not included in CRS management guidelines (105, 106). In a case report, Xiao et al. reported that in a 23-year-old male R/R B-ALL patient, after performing TPE along with the administration of dexamethasone, CRS (grade 3) was mitigated and controlled whereas treatment with antiallergic and antipyretic drugs, glucocorticoids, and tocilizumab was not effective against the progressing condition of the patient’s CRS (105). Moreover, another case report has also reported that TPE has been effective in the treatment of CAR-T-induced CRS of a 4-year-old female patient with R/R B-ALL (107). Additionally, the report of a clinical trial (NCT02349698) involving ten patients has indicated that tocilizumab, glucocorticoid, and TPE have been effective in the successful controlling of severe CRS (grade 3-4) developed in four of the patients (108). However, the broader application of this approach for the management of CAR-T-related CRS lies in the outcomes of more comprehensive future clinical studies in this regard.

Hemofiltration ( Figure 2B ) has not been generally utilized as a clinical approach for controlling CAR-T therapy-related CRS. However, studies have used hemofiltration to help patients with high-grade CRS (grade ≥4), with developed acute kidney injury, to recover their renal functionality (109). Moreover, a case report has indicated that hemofiltration has been helpful in mitigating severe CRS (grade 4) and preventing multiple organ failure and pulmonary infection in a B-ALL patient after CD19-redirected CAR-T therapy (110). It is important to state that in this case, tocilizumab and glucocorticoids were not successful in controlling the mentioned adverse events (110). Furthermore, another study has also proposed that continuous renal replacement therapy (CRRT) can be an additional approach for controlling CAR-T therapy-related CRS that is resistant to conventional treatment (111). This study has demonstrated that CRRT is effective in mitigating sepsis which has a similar pathophysiological mechanism to CRS (111). However, as such studies themselves indicate that there are still various unanswered questions in this field that require addressing (111).

Recently, studies have indicated that there is a relationship between CAR-T infusion dose and the occurrence of CRS (112–114). Therefore, a fractionated dosing scheme can retain high response rates with acceptable tolerability in adult R/R B-ALL patients (113, 114). So far, two clinical trials have evaluated this strategy. The first study (NCT01029366) included R/R B-ALL patients alongside patients with other CD19-positive malignancies and the second one (NCT02030847) only included adults with CD19⁺ R/R B-ALL (113, 114). In detail, Frey et al. administered tisagenlecleucel to adult R/R B-ALL patients after lymphodepletion as either a one-time infusion or fractionated infusions split over 3 days (10% on day 1, 30% on day 2, and 60% on day 3) (113). The total planned CAR-T infusion dose in their study varied with adaptive protocol modifications in response to efficacy and CRS occurrence (113). The second clinical trial included 35 patients in 3 different dosing cohorts which included the low-dose cohort (9 patients), the high-dose single infusion cohort (6 patients), and the high-dose fractionated (HDF) cohort (20 patients) (113). In the low-dose cohort, the patients received single or fractionated dosing, which resulted in manageable toxicity with a 33% CR rate (113). In the high-dose single infusion cohort, half of the patients died due to refractory CRS (grade 4/5) and culture-positive sepsis, and the other half achieved CR (113). Moreover, the HDF cohort had a 90% CR rate and manageable CRS (grade ≤3) (113). Of note, according to Penn Grading System for CRS, grade 4 is characterized by life-threatening complications including hypoxia which necessitates the appliance of mechanical ventilation and hypotension requiring the administration of high-dose antihypotensive agents. Also, grade 5 is defined as CRS-caused mortality (113). The HDF scheme resulted in the highest survival rate with a 2-year OS of 73% and event-free survival of 49.5% (113). The researchers of this study proposed that a fractionated dosing scheme of CTL019, for administration to adult R/R ALL patients, with intrapatient dose modifications can optimize the safety of B-ALL CAR-T therapy without impairing its efficacy (113). Additionally, these clinical trials have demonstrated that a fractionated dosing scheme, where day 2 and day 3 doses are held in response to early CRS, enables individualized dose modifications for achieving a better balance of efficacy and safety in comparison with protocols suggesting a defined single-dose infusion (113).

Conditioning regimen, also known as lymphodepleting conditioning regimen, is the occasional use of chemotherapeutic agents in cell-based cancer therapies such as CAR-T therapies (regardless of autologous or allogeneic CAR-T therapies) (116). It has been demonstrated that conditioning regimen is an important factor affecting the clinical outcomes of CAR-T therapy (116). Studies have demonstrated that conditioning regimen prior to adoptive cell transfer significantly enhances the efficacy of therapies with ex vivo-expanded tumor-infiltrating lymphocytes (TILs) (117). In brief, lymphodepletion chemotherapy provides an appropriate environment for infused T cells to expand by helping create a supportive space in the immune system of the recipients (116–119). Lymphodepleting conditioning regimen acts by several mechanisms including eliminating the recipients’ T and B lymphocytes as well as NK cells, the eradication of immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells, inducing co-stimulatory molecules and downregulating indoleamine 2,3-dioxygenase (IDO) in tumor cells, the elimination of homeostatic cytokine sinks such as IL-2, IL-7, and IL-15, and finally promoting the expansion, function, and persistence of the adoptively transferred T cells by eliminating the factors interfering with their activity (117–119).

Fludarabine and cyclophosphamide are two chemotherapeutic agents that are occasionally used as the conditioning regimen for B-ALL patients receiving CAR-T therapy (120, 121). Studies have demonstrated that using the combination of these drugs in the lymphodepleting conditioning regimen would result in improved CD19-redirected CAR-T expansion and persistence, better clinical outcomes, and disease-free survival of the patients in comparison with non-fludarabine lymphodepleting regimens (120, 121). Others have shown a relationship between elevated in-serum IL-15 levels and clinical response after CD19-redirected CAR-T therapy (122). It is also believed that the probability of achieving a favorable cytokine profile is higher in patients who receive high-intensity lymphodepletion in comparison with those that receive low-intensity lymphodepletion (123). This occurrence in turn is associated with better clinical outcomes (123).

Genome-editing technologies have been utilized for addressing the unfavorable limitations of using allogeneic T cells (124–127). In this regard, Torikai et al. have utilized the zinc finger nuclease (ZFN) genome-editing technique to eliminate the expression of the endogenous αβ TCR in allogeneic CD19-redirected CAR-Ts (128). They demonstrated this method can prevent allogeneic CAR-T-mediated GvHD without compromising CAR-T functionality or antitumor activity (128). Stepping further, they used the same genome-editing method and disrupted the expression of HLA-A in CD19-redirected CAR-Ts, and demonstrated that this strategy can protect CAR-Ts from lymphodepletion therapy, alongside eliminating the possibility of GvHD (129).

Other researchers have developed practical platforms to demonstrate the applicability of the TALEN genome-editing technique for the production of “off-the-shelf” allogeneic CAR-Ts (130–132). In this regard, Poirot et al. used TALEN for the simultaneous disruption of two genes in allogeneic CAR-Ts (130). First, the researchers disrupted the TCRα constant (TRAC) gene expression which resulted in the elimination of TCRαβ expression; therefore, it abrogated the potential of the allogeneic CAR-Ts for GvHD mediation (130). The second knock-out was aimed at rendering the universal allogeneic CAR-Ts resistant to destruction by a lymphodepleting or immunosuppressive agent (130). CD52, as the second knock-out target, can be specifically targeted using the lymphodepleting mAb alemtuzumab, thus enabling the lymphodepletion of recipient(s) and the creation of a receptive environment concurrently or before the administration of the TCR/CD52-knock-out CD19-redirected CAR-Ts (130). Poirot et al. demonstrated that the TCR/CD52-knock-out CD19-redirected CAR-Ts were capable of efficient elimination of CD19⁺ tumor targets even while accompanied by the chemotherapeutic agent (130). Taken together, this GMP-compatible scalable process can be considered as a general platform for the manufacturing of “off-the-shelf” CAR-Ts from the T cells of healthy third-party donors (130). Similarly, Qasim et al. also generated allogeneic TCR/CD52-knock-out CD19-redirected CAR-Ts from non-HLA matched donors, and demonstrated that these genome-edited universal allogeneic CAR-Ts did not mediate alloreactivity in infant recipients with R/R B-ALL (132). They also reported that these CAR-Ts mediated molecular remission in the recipients with acceptable persistence while being anti-CD52 therapy-resistant (132). Moreover, other researchers have also demonstrated that simultaneous knock-out of TCRαβ expression and deoxycytidine kinase (dCK) can result in the production of allogeneic CD19-redirected CAR-Ts that do not mediate GvHD and are resistant to dCK phosphorylation-dependent lymphodepleting regimens (131).

It is worth mentioning that the results of two Phase I trials using TCR-knockout CD19-redirected CAR-T therapy in both children and adults (NCT02808442 and NCT02746952, respectively) have demonstrated the practicability of using allogeneic genome-edited CAR-Ts for the treatment of patients with R/R B-ALL (133). The results of such trials indicate that allogeneic CAR-Ts can mediate acceptable tumoricidal activity with manageable levels of toxicities and adverse events (133). Such CAR-Ts can be beneficial for the treatment of heavily pretreated R/R B-ALL patients with rapidly progressing diseases since autologous CAR-Ts may not be accessible in these cases in terms of meeting the adequate cell number or final-product quality (133).

In addition to the abovementioned strategies, CRISPR-Cas9-based genome-editing method can also be utilized for the generation of universal allogeneic CAR-Ts (134–137). One study has used CRISPR-Cas9 to selectively insert the CD19-redirected CAR transgene into the TRAC locus (134). This mechanism results in the deficiency of the endogenous TCR expression and it ameliorates in vivo CAR-T functionality while reducing the risk of alloreactivity as compared with retrovirus-mediated random CAR transgene insertion (134). It also postpones the effector T-cell differentiation and exhaustion of CAR-Ts and facilitates the efficient internalization and consequent re-expression of CAR molecules upon multiple target antigen engagement (134). MacLeod et al. made a similar attempt and demonstrated that their TCR-knock-out CD19-redirected CAR-Ts exhibited superior antitumor activity in preclinical mouse models without mediating GvHD (136). Furthermore, researchers incorporated multiple gRNAs into a CAR-encoding lentiviral vector which resulted in the simultaneous knock-out of the endogenous TCR and Beta-2 microglobulin (b2M) of HLA class I, thereby paving the way for a practical strategy for the production of universal allogeneic CAR-Ts (137).

More recently, Georgiadis et al. demonstrated that incorporation of an sgRNA element into the Delta U3 3′ long terminal repeat (LTR) of their CAR-encoding lentiviral vector would result in the production of a self-inactivating lentiviral terminal vector that combines CAR expression with CRISPR-Cas9 effects (135). In detail, after transcription and duplication of the hybrid DU3-sgRNA and electroporation-facilitated delivery of Cas9 mRNA, this platform cleaves the TRAC locus and enables enriching the TCR-knock-out CAR-T population using automated magnetic separation (135). These CAR-Ts would retain their CAR-associated specificity and antileukemic activity, have superior persistence as compared with their conventional counterparts, and would not mediate alloreactivity in the respective recipients (135). However, despite what we have discussed, a recent study by Stenger et al. has indicated that CD19-redirected CAR-Ts harboring endogenous TCR have superior persistence and mediate considerably prolonged leukemia control in vivo as compared to their CRISPR-Cas9-mediated TCR-knockout counterparts (138). Conclusively, it can be mentioned that more clinical information is required for drawing more substantiated conclusions.

VSTs that harbor their native TCRs can be used as a source of universal allogeneic T cells in CAR-T therapy (139, 140). Tabelecleucel (tab-cel^®) is a universal allogeneic T-cell immunotherapy using Epstein-Barr virus (EBV)-specific T cells that harbor TCRs specifically targeting EBV antigens (139, 140). It has been demonstrated that CD19-redirected CAR-Ts generated using EVB-specific T cells are well-tolerated with poor mediation of GvHD and CRS suggesting a promising approach for generating universal allogeneic CAR-Ts (140).

VSTs are now being investigated for the prevention of viral infections in recipients of hematopoietic stem cell transplantation (HSCT) since these cells can reconstruct the recipients’ impaired immune system and confer immunity against life-threatening viral infections after HSCT without GvHD mediation (141, 142). This feature of VSTs has encouraged researchers to use them as a reliable source for the generation of allogeneic CAR-Ts (142, 143). An ongoing Phase I clinical trial (NCT00840853) has studied VSTs genetically engineered to express CD19-redirected CARs to see if these cells can maintain their antiviral activity alongside exhibiting specific tumoricidal functionality in patients with CD19⁺ B-ALL after HSCT (142). According to the results of this clinical trial so far, the investigators stated that these CAR-Ts induced no infusion-related toxicities (142). The researchers have also added that vaccinating the recipients of these CAR-Ts with viral antigens may result in enhanced CAR-T expansion and antitumor activity (142). However, this clinical trial is planned to be completed in 2031; therefore, more accurate clinical outcomes can be obtained by then.

Lately, hiPSC-derived CAR-Ts have gained a great deal of attention as universal allogeneic CAR-Ts (144, 145). In detail, peripheral blood-derived T cells are converted to hiPSCs and genetically engineered to express the CD19-redirected CAR with targeted integration into the TRAC locus followed by clonal selection carried out for generating a master hiPSC line, termed TRAC-TiPSC (144, 145). These cells are also genetically manipulated to express a high-affinity form of CD16 with proteolytic resistance for addressing tumor antigen escape (144, 145). The mentioned characteristics of hiPSC-derived CAR-Ts render them incapable of GvHD mediation (144, 145). Taken together, the researchers of these studies indicated that since such CAR-Ts mediated encouraging preclinical results as an effective and safe allogeneic product, it might be feasible to evaluate these CAR-Ts in a Phase I clinical trial in patients with B-cell neoplasms (144, 145).

Recently, researchers have demonstrated that T cells isolated from postpartum human placenta/umbilical cord blood can be used for the generation of CD19-redirected CAR-Ts, termed P-CD19-redirected CAR-Ts (146). P-CD19-redirected CAR-Ts are mainly naïve (CD45RA⁺) T cells and retain their naïve/memory marker expression and harbor a lower expression of effector/exhaustion markers as compared to peripheral blood mononuclear cell-derived CAR-Ts (146). Moreover, these cells also have superior immune tolerance to HLA mismatch and exhibit weakened allogeneic activation resulting in a lower GvHD occurrence possibility (146). Researchers have suggested that these features of P-CD19-redirected CAR-Ts make them a promising candidate for universal allogeneic CAR-T therapy (146). It is worth mentioning that the endogenous TCR expression of these T cells can be abrogated using the CRISPR genome-editing technique to minimize the risk of endogenous TCR-mediated GvHD (146).

One strategy for overcoming the issue of cellular immune rejection is the abrogation of immune responses mediated by both activated alloreactive T cells and NK cells that direct the elimination of foreign cells through different mechanisms (147–151). Mo et al. have developed an engineered receptor called ADR that mediates the depletion of activated host T and NK cells through selective recognition of the cell surface receptor 4-1BB since this molecule is temporarily upregulated on the cell surface of activated CD4⁺ T cells and CD8⁺ T cells, as well as NK cells (151). The 4-1BB-specific chimeric ADR is composed of a 4-1BB-recognizing fragment derived from 4-1BB ligand (4-1BBL) that is connected to the intracellular CD3ζ chain through a spacer and a TM region (151). In vitro and in vivo results have indicated that ADR-expressing T cells manage to resist cellular rejection by targeting alloreactive lymphocytes and, on the other hand, they manage to spare resting lymphocytes (151). Furthermore, Mo et al. co-expressed ADR alongside second-generation CD19-redirected CARs, and demonstrated that T cells co-expressing these two chimeric receptors can preserve their independent anti-rejection and antitumor functionality (151). Moreover, T cells expressing only CD19-redirected CARs and T cells co-expressing both CD19-redirected CARs and ADRs have exhibited similar cytotoxic profiles against CD19⁺ cells, according to Mo and colleagues (151). Conclusively, these researchers have proposed that co-expression of CARs and ADRs can mediate sustained tumor eradication and produce long-term therapeutic benefit in immunocompetent recipients with hematologic malignancies alongside enabling the generation of rejection-resistant off-the-shelf allogeneic CAR-Ts (151).

Using HLA-matched or HLA-haploidentical allogeneic CAR-Ts can lead to different clinical outcomes in B-ALL CAR-T therapy (106). Researchers have investigated the use of HLA-matched or HLA-haploidentical allogeneic CD19-redirected CAR-Ts (M-CAR-Ts and H-CAR-Ts, respectively) for the treatment of R/R B-ALL patients to evaluate which one can result in better clinical outcomes (106). Recently, Jin et al. described the first-in-human application of M-CAR-Ts in R/R B-ALL patients before allogeneic hematopoietic stem cell transplantation (allo-HSCT) (106). The results of this investigation demonstrated that using M-CAR-Ts for the treatment of R/R B-ALL patients results in higher CR rates but it also leads to more severe toxicities in comparison with using H-CAR-Ts (106). These researchers also reported that the mild infusion-related toxicities occurring in patients receiving H-CAR-Ts enabled using higher dose infusions in these patients in comparison with the patients receiving M-CAR-Ts (106). It is important to mention that even though these researchers did not observe GvHD or any uncontrolled infusion-related toxicities, there is still a need for paying considerable attention to GvHD and other infusion-related toxicities (106). Other researchers have also investigated the use of H-CAR-Ts (152–154). In particular, Cai et al. and Zhang et al. have reported that using H-CAR-Ts has beneficial therapeutic effects as part of a conditioning regimen for allo-HSCT (152, 153). However, it is suggested that, as compared to M-CAR-Ts, the lower efficacy of H-CAR-Ts may be due to their heterogeneity in mediating graft rejection (106). Additionally, it is yet to be discovered that whether using a stronger immunosuppressive treatment before the infusion of H-CAR-Ts can enhance the efficacy and antitumor activity of these cells (106).

Different investigations have demonstrated that human CD4⁺ T cells and CD8⁺ T cells consist of functionally and transcriptionally separate subsets with varied in vivo proliferation and persistence capacities after ex vivo expansion and adoptive transfer (172–177). Sommermeyer et al. have shown that human CD19-redirected CAR-Ts manufactured from purified CD8⁺ or CD4⁺ central memory T (T_CM) cells or naïve T (T_N) cells are more effective in attacking and killing CD19⁺ tumors in immunodeficient mice in comparison with CD19-redirected CAR-Ts manufactured from effector memory T (T_EM) cells (178). Other studies have also demonstrated that in comparison to T_EM cells, CD8⁺ T_CM cells harbor stemness potency alongside showing superior persistence after adoptive transfer (172, 179). This fact proposes the advantages of this subset for providing long-term persistence (172, 179). Moreover, in the first clinical trial assessing the feasibility of selecting and engineering defined T-cell subsets, Turtle et al. conducted a Phase I/II clinical trial to evaluate CD19-redirected CAR-Ts manufactured from separately modified defined CD4⁺ T-cell and CD8⁺ T-cell subsets (180). These researchers administered this product in a defined CD4⁺:CD8⁺ composition and in a dose-escalation/de-escalation format to adult B-ALL patients after lymphodepletion chemotherapy (180). They found that the infusion of CAR-T comprised of a uniform ratio of CD4⁺:CD8⁺ shows a correlation between cell dose and earlier and higher peak expansion of clonally diverse CAR-Ts (180). These findings had not been reported in other investigations in which CAR-Ts were manufactured and infused without the consideration of the CD4⁺:CD8⁺ ratio (180). Their results also demonstrated that the infusion of a defined ratio of CD19-redirected CAR-Ts manufactured from CD8⁺ T_CM cells and CD4⁺ T cells could provide a synergistic improvement in potency (180). Also, the results of preclinical investigations in Raji tumor-bearing immunodeficient mice receiving CAR-Ts manufactured from distinct T-cell subsets have shown that when either CD8⁺ CAR-Ts or CD4⁺ CAR-Ts were excluded from the formulated product, the potency of the infused product was significantly reduced (178). It is safe to say that selecting a defined subset of T cells for CAR-T manufacturing with a uniform composition may provide reproducible potency in clinical therapy (180). Moreover, Biasco et al. have recently highlighted the impact of different clonal subtypes of T cells in the final CAR-T product on early antitumor responses and long-term disease-controlling capability of CAR-T therapy (181). These researchers stated that even though T memory stem cells (T_SCM) have a low frequency in the final generated CAR-T product, these T-cell clones significantly contribute to the circulating CAR-T pools throughout both early proliferation and prolonged disease-controlling persistence (181). As suggested by Biasco et al., these findings underscore the significant role of T_SCM in both early anti-leukemic CAR-T reactions and prolonged disease control (181). However, it is important to mention that CAR-T prolonged persistence, after achieving the desired clinical outcomes and disease control, might lead to cytopenia which should be taken into consideration (182).

The different cell culture media formulations used for the activation and expansion of T cells ex vivo, and eventually generating CAR-Ts can be optimized in many ways since they tend to have significant impacts on various aspects of the final T-cell product (183, 184). The cell culture media formulations currently used are dependent on fetal bovine serum (FBS) or human serum (HS) (183, 184). Researchers have investigated the effects of the human transfusion-grade whole blood fraction-derived concentrated growth factor extracts, named Physiologix™ xeno-free (XF) hGFC (Phx), on CAR-T expansion and function (183, 184). This method has been developed to resolve the supply issues of FBS or HS (183, 184). It has been found that Phx enhances T-cell proliferation in both research and clinical-grade media and improves lentiviral-mediated gene transferring and expression (183, 184). Moreover, CAR-Ts expanded ex vivo in Phx-conditioned media harbor advanced in vivo expansion and antitumor activity as compared to those of CAR-Ts expanded ex vivo in HS-conditioned media (183, 184). Phx also enhances the number of naïve T cells (CD45RO^-/CCR7⁺) and central memory T cells (CD45RO⁺/CCR7⁺) in both CD4⁺ T-cell and CD8⁺ T-cell populations which contributes to higher persistence and durability of CAR-Ts resulting in better clinical outcomes (183, 184). Geiger and colleagues have also found that, upon T-cell activation, significant changes occur in the arginine metabolism resulting in a decline in the concentration of the intracellular L-arginine (185). Therefore, these researchers demonstrated that supplementing T-cell culture media with L-arginine can increase the reduced intracellular L-arginine levels in activated T cells and it can induce oxidative phosphorylation instead of glycolysis (185). This effect can result in the central memory phenotype development (185).

The methods currently used for generating FDA-approved CAR-Ts include using anti-CD3 and IL-2 or anti-CD3 and anti-CD28 beads (186). However, these methods result in the generation of CAR-Ts expressing exhaustion markers with an effector phenotype (186). Studies have demonstrated that using IL-7 and IL-15 during the ex vivo culturing of T cells would result in the production of CAR-Ts with T memory stem cell phenotype (CD45RA⁺/CCR7⁺) that exhibit superior tumoricidal activity, expansion, and persistence in vivo as compared with their counterparts expanded ex vivo using IL-2 (187). Others studies have also reported very similar results alongside adding that using IL-7 and IL-15 during the ex vivo culturing of T cells also elevates the response rate of the resultant CAR-Ts to anti-PD-1 adjuvant therapies which is mainly due to the anti-PD-1-responsive CD8⁺/CD62L⁺/TCF7⁺/IRF4^– population of the CAR-Ts (188). However, Alizadeh et al. have indicated that T cells expanded with IL-15 preconditioning maintain a less differentiated stem cell memory phenotype (CD62L⁺/CD45RA⁺/CCR7⁺) with reduced levels of exhaustion marker expression in comparison with T cells preconditioned with IL-2. They have also stated that using IL-7 or IL-21 alongside IL-15 decreases the favorable influences of IL-15 in the induction of CAR-T phenotype and tumoricidal activity (189).

Additionally, it has been demonstrated that using human platelet lysate (HPL) as a supplement in the culture media of T cells results in a higher number of T cells with central memory phenotype in comparison with human AB serum (ABS)- or FBS-supplemented culturing media (190). It has also been discovered that the presence of various cytokines, such as IL-7, in HPL is responsible for the phenotypic differences observed in the mentioned study (190). Furthermore, Torres Chavez et al. have also obtained similar results indicating that HPL-supplemented ex vivo culturing media support T cells to preserve their less differentiated cell phenotype resulting in their longstanding antitumor activity and persistence (191). Above all this, some researchers have demonstrated that treatment of mouse CD8⁺ T cells with the S enantiomer of the 2-hydroxyglutarate (S-2HG) in ex vivo culture significantly enhances their in vivo expansion, persistence, and tumoricidal activity (192). Recently, the same group has indicated that S-2HG acts as an immune metabolite and helps clinical-grade allogeneic CAR-Ts maintain their central memory phenotype and exhibit enhanced antitumor activity (193).

According to the relationship between the state of T-cell differentiation and the potential for in vivo engraftment and persistence, some researchers have proposed that reducing the duration of ex vivo culturing can limit differentiation and significant loss of stemness and enhance the efficacy of CAR-T therapy (194). Ghassemi et al. have demonstrated that reduced culturing duration helps CAR-Ts have improved ex vivo expansion and effector function which are directly correlated with enhanced in vivo engraftment and tumoricidal activity (194). These researchers also demonstrated that these CAR-Ts eradicated human B-ALL in a murine xenograft model even at a 6-fold lower dose as compared with their counterparts expanded with the conventional ex vivo durations (194). In a nutshell, Ghassemi et al. demonstrated that the antileukemic activity of CAR-Ts is inversely correlated with their ex vivo culturing duration (194).

Upon activation, T cells steer their metabolism from fatty acid oxidation to glycolysis which helps them keep their effector function (195). It has been found that T cells with a memory-like phenotype have a low level of glucose uptake in comparison with T cells with an effector-like phenotype (195). Sukumar et al. have demonstrated that activating CD8⁺ T cells in the presence of 2-deoxyglucose, which is a glycolysis pathway suppressor, can result in the formation of memory T cells (195). Moreover, other researchers have shown that pharmacologic inhibition of the serine/threonine kinase Akt promotes the expansion of TILs with transcriptional, metabolic, and functional features of memory T cells (196). Furthermore, Urak et al. also highlighted the Akt pathway involvement in T-cell differentiation state and memory phenotype formation (197). They demonstrated that the inhibition of Akt using an Akt inhibitor during ex vivo expansion of CAR-Ts leads to the production of CAR-Ts expressing higher levels of CD62L and CD28 as compared with CAR-Ts generated without Akt inhibition (197). Moreover, these researchers reported that ex vivo Akt-inhibited CAR-Ts exhibit superior antitumor functionality both in vivo and in vitro as compared with conventional CAR-Ts (197).

Lately, it has been found that the differentiation fate of CD8⁺ T cells is regulated by the methylcytosine dioxygenase ten-eleven translocation 2 (TET2) (198, 199). TET2 loss results in the formation of memory-like phenotype in CD8⁺ T cells without any impairment of their expansion or effector function (198, 199). Moreover, TET2-disrupted CAR-Ts have exhibited central memory phenotype at the expansion peak, which highlights the beneficial impact of TET2 suppression in CAR-Ts (198, 199). Furthermore, Kondo et al. have demonstrated that activated effector T cells can be converted into T_SCM-like (iT_SCM) cells by co-culturing with OP9 stromal cells expressing a Notch ligand such as Delta-like 1 (200–202). It has been found that using IL-7 and IL-15 can significantly be beneficial for the production of iT_SCM cells (201). These researchers also indicated that iT_SCM cells are tolerance-resistant alongside exhibiting robust engraftment, persistence, and tumoricidal activity in vivo (202). More recently, Kondo et al. reported that conventional human CAR-Ts can also be converted into iT_SCM CAR-Ts via Notch signaling-mediated mitochondrial metabolic reprogramming (203). Additionally, they found that Forkhead box M1 (FOXM1) is involved in the downstream signaling of the Notch pathway (203). FOXM1 is mainly responsible for the metabolic reprogramming of T cells and stem cell memory phenotype induction, and iT_SCM CAR-Ts generated using FOXM1 induction are as functional as iT_SCM CAR-Ts generated using Notch induction (203). In addition to the mentioned strategies, it is known that the 4-1BB signaling promotes T-cell central memory phenotype formation and preservation in CAR-Ts by promoting oxidative metabolism (204). Therefore, using the 4-1BB co-stimulatory domain in the construct of CAR-Ts helps preserve their central memory phenotype and in vivo expansion capacity (46).