Henceforward, two kinds of CTT are common: CRS and neurotoxicity. The exact mechanism of the pathophysiology of CTT is yet to be demarcated. However, studies have identified a significant mechanism involving an interaction between macrophages and T cells, leading to the release of multiple cytokines and oxides in murine models (19). Subsequently, it was suggested that the infusion of CAR T cells following macrophage depletion could be a promising approach to eliminate CRS (20). Although this depletion increases the risk of infections, the complications can be managed using IL-6 or corticosteroids or a combination of both (21). Recently, another study showed a positive correlation between CRS and hematological toxicities (22). Endothelial activation is a potent member in CRS pathology (23) as supported by a cohort of 133 B cell malignant patients who received CD19 CAR T cell infusion in a dose-dependent manner. In total, 70% of patients developed CRS with variable severity indices (24). Not just this—endothelial activation also disrupts the blood–brain barrier (BBB) in patients with neurotoxicity as warranted by the magnetic resonance imaging results of severe cases (23). A mechanistic approach was developed in a study that showed that CAR T cell products such as tisagenlecleucel responded by the expansion of proliferative memory-like CD8 clones, while another product, axicabtagene ciloleucel responders, displayed more heterogeneous populations. The latter non-responders had elevations in CAR T-regulatory cells, which suppressed conventional CAR T cell expansion and drove late relapses in an in vivo model (25). One of the recommendations, in the context explained above, is reinfusion of CAR T cells along with a combinatorial use of IL-6 and steroids inclusive of former macrophage depletion. Nevertheless, there are various other mechanisms of pathology of CTT, which are potent to grab the attention of researchers, yet careful monitoring and personalized treatment plans are warranted to manage the potential adverse reactions and ensure the safety of the patients.

Currently, some strategies have been reported for the mitigation of CTT; however, further understanding of the exact approach will lead to the precise development of CARs. Researchers are focusing on the preemptive mitigation of CTT in combination with various immunosuppressors which have been proven to be effective devoid of any attenuation of CAR T efficiency—for instance, a clinical study confirms no negative impact on the antitumor activity of CAR T cell therapy when combined with tocilizumab and/or steroid administration and supports no expansion and persistence of CD19-targeted CAR T cells (26). Following the conclusion, a prospective study evaluated the efficacy of tocilizumab to mitigate CRS in 70 patients divided into two groups of high and low tumor burden. The overall response rates (87% in high-tumor-burden and 100% in low-tumor-burden patients) were good enough to support the very strategy as an effective one. The clinical trial successfully met the primary and final goals of the study without any attenuation of the antitumor activity of CAR T cell therapy (27). Dexamethasone—a glucocorticoid, has also been of interest in the mitigation of neurological toxicities due to its exceptional penetration into the central nervous system as per the guidelines of the National Cancer Institute. IL-7 receptor alpha is a well-recognized facet needed for CAR T cell persistence and memory T cell formation (28). In this case, dexamethasone has been shown to upregulate IL-7 receptors to enhance CAR T cell persistence and antitumor activity (29). Urak and colleagues suggest that the effect of dexamethasone is not only restricted to specific T cell subsets while the same upregulation was also observed in peripheral blood mononuclear cells (PBMCs), naïve cells, memory T cell-derived CAR T cells, and untransduced T cells. The study does not affect the efficacy of CAR T cells in vivo or in vitro. These facts are promising in understanding the mechanism of dexamethasone in CAR T cell therapy. When combined with ruxolitinib, dexamethasone synergistically reduced the disease symptoms in a murine model. Mechanistically, JAK-dependent cytokines IL-2 and IL-12 conferred resistance to dexamethasone, but not etoposide, and ruxolitinib attenuated STAT5 activation to enhance dexamethasone-mediated cell death in the presence of IL-2 or IL-12 (30). Other various drugs are under investigation to be used in combination with CAR T cell therapy. In this text, we advocated the inhibition of CTT utilizing reported cytokines and combining various immunosuppressants and steroids. These drugs reveal an in-depth understanding of the mechanism of mitigation of CTT, including CRS and neurotoxicity. Further research in this area is de rigueur to proceed with the mitigation of CTT for effective CAR T cell therapy.

Mechanistically, chemokine-transforming growth factor β (TGF-β) activates tumor-associated fibroblasts, which, in return, upregulates the extracellular matrix proteins to T cell infiltration into solid TME (31). Interestingly, a direct restriction to CAR T cell infiltration by TGF-β has also been observed, whereby TGF-β not only downregulates CXCR3 expression but also increases the binding of Smad2 to CXCR3 promoter (32). The search for effective and safer therapeutics targeting the TGF-β pathway is further complicated by various functions that TGF-β performs in normal tissues (33). We accordingly advocate further extensive research to investigate the contextual role of TGF-β for immunosuppression in TME. Rather than TGF-β, several other cytokine and chemokine profiles recruit T-reg cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages and thus limit CAR T cell infiltration. T-reg cells secrete immunosuppressive cytokines and downregulate antigen-presenting cells via cytotoxic T lymphocyte antigen 4, thus preventing T cell activation (34). The immune-suppressive effect of MDSCs also has an unfavorable effect on CAR T cell therapy (35). Krishnamoorthy et al. reviewed a detailed mechanism of suppression of T cells mediated by MDSCs (36). They stated that, generally, MDSCs suppress the immune response by inhibiting the functions of T cells in four distinct pathways as shown in Figure 1A . In this way, we recommend utilizing the drug which could be capable of blocking the MDSC functions within the solid TME. In this aspect, the delivery systems must be capable of targeted unloading of cargo. We also suggest that CAR T cells should be engineered genetically to express molecules that specifically target and eliminate MDSCs within the TME. This recommendation is interestingly supported by a study in which the authors developed CAR T cells with co-expression of receptors for MDSCs. The study provides an evidence of superior anti-tumoral activity of CAR T cells which also improved T cell proliferation in solid TME (38).

Once the minute, safe, and appropriate tumor-associated antigens (TAAs) are determined in the development of CAR T cell therapy, it becomes crucial to address tumor antigen heterogeneity in the subsequent step, as it may be expressed variably in both tumor and normal cells (39, 40). Dana H et al., in 2021, systematically summarized various antigens, targeted in different cancers (41), showing promise in the mitigation of tumor heterogeneity. An interesting study employing a combinatorial therapy demonstrated a compatibility between CARs, T cell receptors (TCRs), and hnCD16 in alleviating tumor heterogeneity (42). This technique is likewise effective for resistant tumors owing to antigen escape. By computational modeling, cellular interactions were examined, and it was thus concluded with a suggestion to employ combinatorial therapy for effective treatment (43). As mentioned above in the mitigation of CTT, the presence of IL-12 enhances the synergistic effect of steroid therapy in combination with ruxolitinib. This is an evident boon that it is present in solid TME (44). We hereof recommend a tumor-specific condition employing the IL-12-mediated expression of CARs. Nevertheless, investigations are warranted to inspect off-tumor toxicity along with IL-12 dosages. A similar study was conducted aiming to augment tumor specificity (45). NK cells detect tumor ligands through NK receptors, including NKp30. A subset of CD8+ T cells expressing NKp30 recognizes B7H6, a tumor ligand frequently overexpressed in cancers (46). Apropos of this research, Correia et al. (2021) latterly engineered a dual recognition strategy in the form of innate-like NKp30+CD8+ T cells with a combined expression of TCRs and CARs, thus successfully targeting tumor heterogeneity (47). Furthermore, novel criteria for the conscription of CAR T cell therapy, including preselection for the intensity and proportion of targeted antigen, are warranted. Although prescribing the definite criteria could be tricky, hereto we suggest concurrent or in combination targeting of different tumor antigens, which is promising in leading to a more effective antitumoral effect and possibly reducing the emergence of tumor variants that lack the targeted antigen.

Generally, upon tumor initiation, normal and tumor cells upregulate inhibitory signaling pathways, driving the tumor progression and its immune escape. Blockade therapy has shown to reverse this suppression and has shown promising effectiveness in hematological malignancies (48). Henceforth, the use of immune checkpoint inhibitors, specifically PD-1 blockade, has been under investigation. Chronic antigen stimulation in tumor sites leads to inhibitory receptor upregulation and CAR T cell exhaustion in a PDL1-dependent manner (49). Blocking the PD-1:PDL1 interaction using mAbs can rescue exhausted CAR T cells as shown in Figure 1B , but multiple administrations are required due to the short half-life of antibodies and immune-related adverse events (50). Localized immune checkpoint inhibition at the tumor site using modified CAR T cells secreting PD-1 blocking scFv can increase the killing activity and restore the cytotoxic activity in solid tumors. The very strategy has shown a positive response in CAR T cell exhaustion in vivo that indicates a promising synergistic effect with improved efficacy (51–53). Genetically modified CAR T cells are also under investigation for local inhibition of immune checkpoints incorporating specific mechanisms—for example, engineered CAR T cells expressing a dominant-negative receptor can disrupt the signaling of immune checkpoint molecules. Genetically modified CAR T cells also secrete blocking single-chain variable fragments, which can prevent immune checkpoint molecules from binding to their targets. Lastly, CAR T cells can be engineered to interfere with intracellular signaling pathways by disrupting the gene expression as depicted in Figure 1C (54). By implementing these innovative techniques, it is anticipated that CAR T cell therapy can overcome immune checkpoint-mediated suppression and offers enhanced therapeutic outcomes for cancer patients.

MDSCs can upregulate checkpoint molecules like CTLA4, T cell immunoglobulin, and mucin domain-containing protein 3 (TIM3) on T cells, leading to T cell apoptosis through Fas signaling (55). Another study focuses on targeting B7-H3 immune checkpoint in non-small cell lung cancer which shows promise in T cell activation and target cell death. The study also finds that anti-B7-H3 blockade has a role in altering glucose metabolism through reactive oxygen species-mediated pathways (56). Moreover, rebalancing the TGF-β1/BMP signaling pathways shows promise in exhausted T cells and induces higher antitumoral targeting while synergizing the immune checkpoint blockade (57). This data may provide better insights into solid TME for effective tumor-killing activity.

A careful selection of tumor-specific antigens is warranted as they are rare and require precise identification following the use of TAAs. The main issue is the heterogeneity of tumor-specific antigens and their expression on normal, non-tumoral cells vulnerating them for off-tumor toxicity. Generally, the antigens targeted by CAR T cells are tumor-associated, rather than tumor specific. While these antigens may be highly expressed within the solid tumor microenvironment, it is important to note that normal cells also express a small amount of these antigens (58).

Recent developments in the field of CAR T cell therapy have led to the emergence of novel technologies which hold significant potential in addressing the challenges of on-target/off-tumor toxicity. Researchers have developed novel technologies like ZAP-70-based logic-gated intracellular network CARs through the application of Boolean logic gating and the engineering of intracellular T cell signaling molecules (59). This fact demonstrates superior efficacy and safety while bypassing upstream signaling proteins. The utilization of logic gating circuits and synthetic biology in CAR T cell engineering has shown promising specificity in preclinical mouse models (60). However, the translation of these approaches into clinical studies is yet to be fully explored and evaluated. Furthermore, SynNotch CAR utilizes synthetic Notch receptors in mitigating the toxicity by enhancing the specificity and functionality (61). It allows enhanced selectivity via improved discrimination between tumor cells and healthy tissues, minimizing off-target toxicity (62). The activation of SynNotch CAR is likewise programmable by enabling downstream signaling pathways and expressing effector molecules to offer enhanced flexibility (63). Notably, it also allows fine-tuning of immune responses by incorporating inducible signaling domains or regulatory elements that modulate CAR-T cell activity, promoting controlled and adaptable immune responses (64). Another important development is the T cell redirected for universal cytokine killing (TRUCK) CAR which combines the killing ability of CAR with the production of therapeutic cytokines (65) such as inducible IL-18 (66). There are conditional cytokine expression systems that enable controlled cytokine release, reducing the risk of off-target effects and cytokine-related toxicities (67, 68). Importantly, TRUCK CARs allow antigen-specific targeting minimizing off-target toxicity (69, 70).

Specifically, apart from the above-mentioned developments, gene editing practice is more promising in this research area. To enhance T cell responses to antigens and decrease exhaustion rates, the very technology has been employed (71). Furthermore, the use of such technologies may allow for the development of T cell populations that can be used in allogeneic applications. Exploiting clustered regularly interspaced short palindromic repeat (CRISPR) in mitigating on-target/off-tumor toxicities is itself another research arena whereby it is used to create CAR resistance to immune suppression (72). Modifying CAR T cells to target tumor-specific antigens using CRISPR is another promising strategy as it has been demonstrated that CRISPR has been used to knock out the expression of the CD19 antigen on normal B cells, allowing CAR T cells to target only tumor cells that express CD19 (72). This approach has the potential to reduce the risk of CTT without targeting normal cells. An extensive review of such modifications via CRISPR technology for CARs (as shown in Figure 2 ) presents encouraging outcomes in clinical and preclinical trials (73). Further research into all these insights is necessary to develop effective strategies for mitigating the risks associated with CAR T cell therapy.

Herein the aforementioned barriers have been on the bull’s eye to steer CARs toward promising efficacy. However, newly emergent evidence suggests novel roadblocks in the form of tumor parenchyma and stromal cells in solid TME (74). These two distinct yet synergistic elements engage in a dynamic cross-talk, thereby facilitating and sustaining a resistant solid TME. Notwithstanding, the very notion not only initiates and promotes tumor growth but also augments the metastatic potential of tumors (75), presenting new research areas in overcoming the roadblocks to CAR T cell therapy. The promising candidates comprise tumor-associated fibroblasts (TAFs) secreting fibronectin and collagen-like proteins. Further cellular entities such as MDSCs, epithelial, endothelial, or mesenchymal cells, adipose cells, and bone marrow-derived mesenchymal cells have also been observed to transit to TAFs (75, 76) as depicted in Figure 3 . Noticing all these entities, CAR T cell therapy acquires a giant barrier to incapacitate. Consequently, the development of innovative strategies and combinatorial strategies with other established treatment modalities are warranted as a promising gateway to CAR T cell therapy research arena.

Accordingly, modulation of TME in overcoming stromal barriers has shown promise. Geng F. et al., in 2019, examined the effectiveness of a DNA vaccine expressing fibroblast activation protein-α (FAP-α) in altering the solid TME in breast cancer (77). In this aspect, CARs that should target FAP-α to mitigate the barrier are needed to be developed, allowing more infiltration and persistence, or else CAR T cell therapy may be combined with the pre-infusion of nanoparticles (NPs) with FAP-specific antibodies cargo as evidenced by Zhen et al. in 2017 (78). There are a lot more options; one of that is targeting pathways of stromal interactions which may be employed as a pre-emptive strategy or combinatorial agents as extensively reviewed by van der Spek et al. in 2020 (76).

By and large, the text herein highlights the potential of CAR T cell therapy and underscores a prerequisite for sustained research and collaboration to optimize its clinical application and offer hope for patients with solid tumors.