Hurdles to breakthrough in CAR T cell therapy of solid tumors

Autologous T cells genetically engineered to express chimeric antigen receptor (CAR) have shown promising outcomes and emerged as a new curative option for hematological malignancy, especially malignant neoplasm of B cells. Notably, when T cells are transduced with CAR constructs, composed of the antigen recognition domain of monoclonal antibodies, they retain their cytotoxic properties in a major histocompatibility complex (MHC)-independent manner. Despite its beneficial effect, the current CAR T cell therapy approach faces myriad challenges in solid tumors, including immunosuppressive tumor microenvironment (TME), tumor antigen heterogeneity, stromal impediment, and tumor accessibility, as well as tribulations such as on-target/off-tumor toxicity and cytokine release syndrome (CRS). Herein, we highlight the complications that hamper the effectiveness of CAR T cells in solid tumors and the strategies that have been recommended to overcome these hurdles and improve infused T cell performance.


Introduction
Autologous T cells redirected by chimeric antigen receptors (CARs) have been emerged as a new weapon for cancer therapy. The idea of constructing CARs by incorporating the single-chain fragment variable (scFv) domain of an antibody with TCR constant domain was first developed in the late 1980s, following the identification of Ig-TCR chimeric proteins in myeloma and human T cell tumors [1]. Engineered T cells are generated in several steps, starting with the collection of leukocytes from the donor's or patient's blood through the leukapheresis, followed by the isolation of T lymphocytes and the use of viral or non-viral vectors for CAR construct transduction [2]. Chimeric antigen receptors are composed of three main parts: an ectodomain derived from the antibody's scFv to detect cancer cells, a transmembrane region for receptor insertion into the plasma membrane, and an endodomain for signal transduction [3]. According to the number and composition of intracellular signaling molecules, CARs are categorized into four generations [2]. The expression of these synthetic molecules in T cells results in antigen recognition and activation of modified T cells in an MHC-independent manner [4].
Tumor cells exploit numerous tactics to counteract tumor-infiltrated lymphocytes (TILs) effector activity, such as downregulation of the molecules involved in antigen presentation and reducing costimulatory signals [5][6][7]. Indeed, MHC independence and employing intracellular signaling domain for antigen detection and CAR T cell activation are two advantages of this technique to circumvent the tumor escape challenge. It is pertinent to outline that CAR T cells detect can almost all forms of the antigens and partially hinder the destruction of healthy tissues in hematological malignancies [8]. Due to the benefits mentioned above and the following information drawn from the literature, CAR T cell therapy has proceeded from basic to clinical studies and generated a great deal of enthusiasm in cancer immunotherapy.
CAR-transduced T lymphocytes, especially anti CD19-CAR T cells, have displayed impressive efficacy in patients with B cell malignancies, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), particularly relapsed/refractory B-ALL, non-Hodgkin lymphoma (NHL), and multiple myeloma (MM) [8][9][10][11][12]. The cell manufacturing process, infusion dose (cell number infused per kilogram), preconditioning regimen, CD4:CD8 ratio, and phenotypic characterization vary between these experiments. Besides, cytokine release syndrome (CRS) has been reported following the administration of specific redirected T cells, with symptoms ranging from mild to life-threatening [13][14][15]. Several lines of evidence have suggested that the severity of CRS is depend on the increased serum levels of inflammatory cytokines (including IFN-γ, TNF-α, and IL-6), as well as the volume of tumor burden [16][17][18]. Hence, administration of corticosteroids or IL-6 receptor blocking antibodies has been used to hamper the infused T cells activity and alleviate the symptoms of CRS [19]. Interestingly, incorporating the suicide gene within the CAR construct serves as a remote control for CAR T cell elimination on demand [20]. Despite the relative effectiveness of transduced-CAR T cells in patients with hematological malignancies, it fails to enable marked anti-tumor response in the treatment of solid tumors [21,22]. This paper recapitulated the challenges posed by CAR T cell therapy in solid tumors, as well as strategies to overcome these hurdles.

Restricted access to tumor cells
Efficient infiltration of T cells into the tumor stroma is a critical step for the anti-tumor activity of infused T cells and the success of cancer immunotherapy. Unlike hematological malignancies, cancer cell accessibility is restricted in solid tumors, and several physical barriers, such as tumor vasculature and extracellular matrix, mainly obstruct infused-CAR T cell penetration to tumor tissue. Accordingly, delineating the factors that prevent T cell trafficking and employing counteracting strategies may influence the outcome.

Extracellular matrix
The extracellular matrix (ECM), as a part of the surrounding stroma of the tumor, is composed of fibrous proteins, glycoproteins, polysaccharides, and proteoglycans [23]. Increased expression and density of ECM components in malignant tissue, particularly overproduction and deposition of hyaluronan and collagen, hampers the penetration of therapeutic agents [24][25][26]. The assessment of T cell migration and localization in tumor stroma elucidated an inverse correlation between T cell infiltration and ECM rigidity, i.e., T cells accumulated in the region with low fibronectin and collagen density [27]. Furthermore, elevated collagen density compromise proliferation and cytotoxic activity of T cells and induced the regulatory phenotype [28]. Given these observations, applying ECM degrading enzymes such as hyaluronidase and collagenase could reduce ECM stiffness and also facilitate the delivery of anticancer agents [29][30][31]. Apart from the tumor microenvironment (TME) suppressive effect in T cell infiltration, the CAR T cell manufacturing strategy may also be designed to downregulate the expression of ECM-degrading enzymes [32]. Owing to these facts, Caruana and coworkers induced heparanase (HPSE) expression in CAR-modified T cells, which improved the ECM degradation capacity and the anti-tumor activity of co-expressed CAR T cells (i.e., expressed CAR and HPSE) in solid tumors [32].

Tumor vasculature
Cancer cells, which are characterized by uncontrolled cell division, require the formation of new blood vessels to obtain nutrients and oxygen. The development of aberrant vasculature, together with the downregulation of adhesion molecules involved in T cell extravasation (under the effect of angiogenic factors like bFGF and VEGF), acts as a physical barrier to T cell penetration into the tumor bed [33,34]. Moreover, the endothelial cells in the tumor microenvironment promote FasL and inhibitory molecules expression (such as PD-L1, TIM3, IDO-1, PGE2, and IL-10) which subsequently suppressed effector T cell activity [34,35]. These properties of tumor blood vessels are dependent, at least in part, on the production of VEGF and the overexpression of its receptors [35]. Therefore, CAR targeting VEGFR1 and VEGFR2 were designed and showed promising results in destroying tumor vessels and reducing tumor cell proliferation by restricting nutrients and oxygen [36,37]. In this regard, simultaneous infusion of VEGFR2-specific CAR T cells and antigen-specific TCR transduced T cells improved the outcome of tumor-specific immunotherapy via increasing the infiltration, persistence, and antitumor activity of tumor-specific T cells [38]. As another option, Santoro and colleagues employed the overexpression of prostate-specific membrane antigen (PSMA) on solid tumor vasculature (but not on the normal endothelium) to selectively target the tumor vessels [39]. The authors suggested that the anti-PSMA CAR-transduced T cells directly destroy tumor endothelium and result in secondary tumor regression [39].
As reported by Lohr et al., incubation of glioblastoma endothelial cells with TGF-β reduced the expression of cell adhesion molecules (VCAM-1 and ICAM-1), led to an impairment in the transmigration of T cells [40]. Several authors have considered the role of chemokine's gradient to counteract these restricting factors and improve the CAR T cell localization in the tumor microenvironment. For this purpose, first they determined chemokines that are produced consistently and increasingly by the tumor and tumor-associated cells and then introduced its corresponding receptor into CAR T cells. For instance, forced expression of CCR2b in GD2-CAR T and meso-CAR T improved the homing of modified CAR T cells in neuroblastoma and malignant pleural mesotheliomas (MPM) by binding to CCL2, the chemokine that is substantially produced by many tumors [41,42]. Moreover, evaluation of chemokines levels in patients with Hodgkin lymphoma (HL) revealed the overproduction of CCL17 and CCL22 chemokines by Hodgkin-Reed-Sternberg (HRS) cells that attracted the T helper 2 (Th2) and regulatory T cells (Tregs) through CCR4 interaction, so participated in the formation of immunosuppressive milieu [43]. Co-transduction of CCR4 and anti-CD30 CAR into the T cells using bicistronic vector also potentiated the migratory and anti-tumor activity of redirected T cells in HL, according to Stasi et al. reports [44]. Likewise, investigations have reported the elevated expression of CXCL1 and CXCL8 in TME of melanoma, and thereby the introduction of the CXCR2 gene strengthened the anti-tumor potency of infused transgenic T cells [45]. Additionally, intratumoral administration of oncolytic virus (OV) armed with the chemokine RANTES as chemoattractant agent and IL-15 as a T cell growth factor, combined with anti-GD2-CAR T cells, was led to similar outcomes (i.e., local accumulation and survival of T cells) in the neuroblastoma tumor model [46].
It is valuable to mention that cell penetration in the solid tumor is more complicated than hematological malignancies, thus need more attention to facilitate engineered T cell trafficking. Zhu et al. proposed a novel nanotechnology strategy to improve the therapeutic efficacy of CAR T cells in solid tumors. Their tumor-specific nanostructure remodeled the TME structure and facilitated CAR T cell penetration into the tumor bed via photothermal and nanocatalytic characteristics [47].

Tumor immunosuppressive microenvironment
Tumor cells recruit immunosuppressive cells such as Tregs, myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs) to establish an immunosuppressive milieu and get around immunotherapy approaches. Indeed, tumor-promoting and immunosuppressive capacity of these cells, along with the metabolic properties of the tumor microenvironment, restricts the anticancer activity of infused CAR T cells (Fig. 1) [48,49]. Functional characterization of tumor infiltrated CAR redirected T cells isolated from tumor-bearing mice up to 40 days after intravenous cell infusion confirmed hypofunctional features of CAR TILs. Moreover, the cytotoxic activity and cytokine production ability of mesothelin-CAR TILs (such as IFN-γ and IL-2 secretion) diminished after exposure to specific antigens [50]. Accordingly, myriad attempts have been conducted to generate more potent cells to withstand the hostile microenvironment of solid tumors [48]. For instance, inducible secretion of inflammatory cytokines such as IL-12 and IL-18, as well as blocking the signaling pathways of immuno-suppressive cytokines like IL-4, IL-10, and TGF-β by transducing dominant-negative receptors and inverted cytokine receptor (ICR) in modified T cells modulated the TME and boosted the CAR-modified T cell efficiency [48,[51][52][53][54][55].Further,, Curran et al. documented that constitutive expression of the CD40L gene in CAR T cells heightened the proliferation, cytotoxic activity, and inflammatory cytokine secretion of T cells. Cancer cell immunogenicity and dendritic cell maturation were also enhanced, thus promoting the immune response activation [56].
On the contrary, oncolytic virotherapy as a novel anticancer approach with promising results uses genetically modified viruses preferentially replicated in tumor cells and finally destroy them. According to Rezaei et al., applying these oncolytic products before CAR T cells infusion in solid tumors reverse the tumor immunosuppressive milieu and reinforces the CAR T cell cytotoxic activity [57]. According to Li et al., reports, combination therapy with oncolytic adenovirus targeting TGF-β signaling pathway and meso-specific CAR T cells, reveal a superior effect on inhibiting tumor growth [58].

Hypoxia in tumor microenvironment
Hypoxia is defined as a state of low oxygen availability that occurs in various physiological and pathological conditions. It is a hallmark of the tumor microenvironment that impacts tumor progression and also modifies treatment outcome in cancer patients [59]. Oxygen deprivation induces the stability and nuclear translocation of hypoxia-inducible factor (HIF), which further regulates gene transcription by binding to hypoxia response element (HRE) regions in the promoter of hypoxia-inducible genes, leading to cell adaptation to environmental changes [60]. On the other hand, TCR activation signals or cytokines produced during the infection and inflammatory process could regulate the synthesis and stability of HIF, which in turn affects the activation and differentiation of T lymphocytes [59,61]. Doedens et al. elucidated that TCR-mediated stimulation of T cells and hypoxia condition in the context of persistent infection or tumor microenvironment increased HIF abundance and effector function of cytotoxic T lymphocytes (CTLs). Subsequently, T cell response is dampened partially by von Hippel-Lindau (VHL) factors to protect the body from the devastating effect of an excessive immune response [62]. Notably, stabilization of HIF1α in T lymphocytes parallels with enhancement in the expression of glycolytic enzymes (such as GLUT-1 as a glucose transporter) and reduction in the rate of oxidative phosphorylation [63]. Therefore, augmentation of HIF levels and activity via hypoxia-dependent and hypoxia-independent pathways serves as a modulator for metabolic pathways and is involved in T cell proliferation, differentiation, and effector activity. As cited, the localization of CAR T cells in the hypoxic tumor microenvironment may influence their appropriate performance and considering the responsible mediators contribute to the generation of a more potent CAR engineered T cell. In this regard, Juillerat et al. designed oxygen responsive CAR by fusing the oxygen-sensitive domain of HIF1α to the C-terminal end of the CAR intracellular domain to limit CAR presentation and activation to the hypoxic milieu and discount the on-target/off-tumor toxicity [64]. Alternatively, overexpression of antigens in hypoxia conditions, such as carbonic anhydrase IX Fig. 1 Tumor immunosuppressive microenvironment. The anticancer function of Infiltrated-CAR T cells is impeded by regulatory cells, immune checkpoint molecules (e.g., PD1 and CTLA-4), and immune inhibitory soluble mediators produced by tumor cells or tumor-associated cells. In addition, the metabolic profile of tumor tissue, especially hypoxia, impacts CAR T cell effector activity in TME (CAIX) expression in glioblastoma, is a promising target for redirecting CAR T cells to recognize and so eradicate cancer cells [65].
As noted by Kawalekar and coworkers, the inclusion of distinct costimulatory domains in CAR construct induced various metabolic pathways (such as oxidative phosphorylation or glycolysis) following antigen stimulation to provide the energy required for lymphocyte differentiation, i.e., short-lived effector cell or longlived memory cells [66]. Hence, selection of appropriate costimulatory domain in CAR architecture potentially assists in adaptation and persistence of infused T cells in an oxygen-deprived tumor microenvironment.
Unfavorable metabolic conditions, such as hypoxia, promote ATP breakdown and inhibition of the adenosine kinase (which phosphorylates adenosine to AMP), resulting in adenosine enhancement [67]. Interaction of hypoxia-derived accumulated adenosine in the tumor microenvironment with specific receptors expressed on T cells surface (i.e., A2AR and A2BR) interfere with TCR signaling and thus inhibits the antitumor activity of T cells [68]. Importantly, the interaction of adenosine and G protein-coupled receptors (GPCR) activates protein kinase A (PKA) that subsequently regulates TCR downstream signaling. Expression of RIAD (regulatory subunit I anchoring disruptor peptide) by CAR T cells prohibits PKA localization in immunological synapse, hence blunts the inhibitory effect of adenosine on the mesothelin-CAR T cell activity, according to Newick et al. [69]. Besides, employing small molecules such as BAY 60-6583 (adenosine A2B receptor agonist) could improve the therapeutic efficacy of CAR T cells by affecting multiple targets [70].
Another drawback of hypoxia is the upregulation of an immune checkpoint PDL1 molecule in a HIF-1α dependent manner, which hampers the functionality of infiltrated T cells [71,72]. Interestingly, abrogation of PD1 interaction with PDL1 by PD1 blockade eventually augmented the A2AR expression on tumor-infiltrated CD8 + T lymphocytes, resulting in enhanced susceptibility to immunosuppression by accumulated adenosine [73]. Therefore, simultaneous targeting of the PD1/PDL1 axis and adenosine A2A receptors (through genetic or pharmacological approaches) could improve T cell performance in CAR T cell therapy [74].

Cancer-associated fibroblasts
Cancer-associated fibroblasts (CAFs) (also known as tumor-associated fibroblasts (TAFs)) are one of the most abundant and influential components in the tumor stroma that play a vital role in tumor progression and resistance to immune response [75]. They not only provide a physical barrier to limits the immune cell accessing the tumor, but also secrete growth factors that promote tumor growth, angiogenesis, invasion, and metastasis (i.e., VEGF and PDGF) [76]. Notably, CAFs suppressed T cell activity in two distinct ways: directly through PD1/PDL1 interaction and secretion of inhibitory molecules (such as TGF-β) and indirectly through inducing immunosuppressive phenotypes in the tumor-associated immune cell, such as macrophage M2 phenotype differentiation and recruitment of MDSCs and Treg cells [76,77]. Because of the essential and supporting role of CAFs in cancer progression, it has been proposed that targeting these cells could improve the efficacy of cancer therapy approaches [76]. In this regard, targeting CAFs through specific surface markers, particularly fibroblast activation protein (FAP), has revealed promising outcomes. In a study of FAP-specific CAR T cells in malignant pleural mesothelioma (MPM) as FAP expressing model, Schuberth and colleagues reported that FAP recognizing CAR T cells substantially destroyed FAP-positive targets in vitro (including mesothelioma cells and fibroblasts) and delayed the tumor growth in vivo evaluation [78]. Furthermore, combining the FAP-specific CAR T cells for stroma targeting with tumor-associated antigen (TAA)-redirected T cells (such as EphA2-CAR T) strengthened the antitumor activity of tumor-targeting T cells [79]. Interestingly CAFs produced the CXCL12 chemokine, which excluded T cells from tumor tissue, therefore interrupting the CXCL12 and CXCR4 interaction facilitated T cell penetration into the tumor bed and elevated the success of immunotherapy approaches [80,81]. Meanwhile, co-targeting the tumor with modified CAR T cells and CXCR4/CXCL12 inhibitors such as AMD3100 and NOX-A12 resulted in higher CAR T cell accumulation in the tumor microenvironment and superior anticancer activity compared to adoptive T cell therapy alone [82].

Presence of immunosuppressive cells
Regulatory T cells The high frequency of Treg cells in cancer with immunosuppressive features such as secretion of IL-10 and TGF-β, as well as competition for IL-2, which is required for the effector T cells proliferation, dampens the functionality of infused T cells [83,84]. Numerous strategies have been recommended for defeating the mechanisms of Treg-mediated immunosuppression, including preconditioning with cyclophosphamide for Treg depletion [85], armored CAR T cells with the secretion of pro-inflammatory cytokines, e.g., IL-12 and 1L-18 [86][87][88][89], incorporating CD28 signaling domain into the CAR construct [90,91], and knocking out the TGF-β receptor in CAR T cells [92]. Integration of the CD28 costimulatory domain into the CAR constitution impacts the cell kinetics and promotes T cell proliferation and production of the pro-inflammatory cytokines (e.g., IFN-γ, GM-CSF, and TNF) [93]. Accordingly, selecting the proper intracellular signaling domains is urgently required to circumvent Treg cell-mediated immunosuppression [94].
Antigen recognition by CAR and activation of an intracellular signaling pathway in redirected T lymphocyte results in IL-2 secretion, which assists Treg survival and expansion. In this context, several groups elucidated that modification of lymphocyte-specific tyrosine kinase (Lck) binding motif in the CD28 domain leads to abrogation of IL-2 expression after CAR engagement [95,96]. However, integrating a 4-1BB costimulatory signal into a modified CAR construct significantly improved CAR T cell expansion by compensating for the deficiency of Lck (and also the lack of IL-2) [96]. On the other hand, RNA-Seq analysis of purified T cells from eight cancer types revealed an elevated expression of 4-1BB on tumor infiltrated Treg cells corresponded to peripheral Tregs. Thus, 4-1BB upregulated expression has been defined as an immune checkpoint signature of tumor Tregs and employed as a specific target for the depletion of Tregs in the tumor milieu, which resulted in tumor regression and increased IFN-γ production by specific TCD8 cells [97]. Indeed, 4-1BB, also known as TNFRSF9 (tumor necrosis factor receptor superfamily member 9) and CD137, is expressed on a wide range of immune cells, such as NK, DCs, neutrophils, and Tregs, so the application of anti-4-1BB mAbs tempers the antitumor immunity [98][99][100]. According to Mardiana et al., combination therapy with anti-4-1BB mAbs and anti-Her2 redirected CAR-T cells diminished the tumor infiltrated immunosuppressive cells frequency (such as Tregs and MDSCs) and enhanced antitumor activity of infused CAR T cells [98].
Overall, as a result of vital role of Treg cells in sustaining the self-tolerance and prevention of autoimmunity, exploiting the strategies that selectively and locally eradicated the tumor Tregs, rather than peripheral Tregs, improves the success of immunotherapy approaches, particularly adoptive CAR-T cell transfer therapy, without severe immune-related adverse events (irAEs).

Tumor-associated
macrophages Tumor-associated macrophages (TAMs) are originated from inflammatory monocytes or M-MDSCs (monocytic myeloid-derived suppressor cells) that are directed to the TME by chemoattractants secreted from cancer cells or tumor-associated cells (e.g., CCL2, CCL5, and CSF1). Furthermore, in some types of malignancies, such as glioma, tissue-resident macrophages with an embryonic origin (i.e., microglia in brain tissue) operate as a component of the TAMs population [101]. These cells contribute to the progression and metastases of tumor cells by secreting soluble factors consisting of growth and proangiogenic factors, cytokines, and proteolytic enzymes [102]. In addition, enhanced TAM polarization toward the M2 phenotype with immunosuppressive features assists tumor evasion from immune surveillance and limits the activity of infused redirected CAR T cells in tumor sites through multiple pathways, including the release of immunosuppressive mediators (such as IL-10, TGF-β, and IDO), the expression of immune checkpoint molecules, and recruiting regulatory T cells [102,103].
As previously reported, TAMs targeting via specific CAR T cells (anti-CD123 CART) or impacting on their polarity by cytokine secreted from modified T cells (such as IFN-γ and GM-CSF) reduced the immune suppression accomplished by these cells [104,105]. Chuang and coworkers have shown that applying the appropriate dose of immunomodulatory agents such as Sorafenib as a tyrosine kinase inhibitor in combination with adoptive T cell therapy modulates the immunosuppressive milieu of TME and enhances the therapeutic effect of transferred T cells [106]. Furthermore, administration of the Sorafenib in combination with the GPC3-CAR T cells revealed that the subpharmacologic dose of Sorafenib induced the secretion of IL-12 from TAMs and subsequently enhanced CAR T cells activity [107].
On the other hand, TLR3 engagement by specific ligand Polyinosinic-polycytidylic acid (Poly I:C) reverts the M2 phenotype of macrophages to the M1 subtype, which was confirmed by increased expression of M1 phenotype markers (i.e., CD40, CD80, and CD86) and reduction in the CD206 expression level on M2a macrophages (as indicator marker for M2 phenotype) [108]. Besides, stimulation of macrophages with TLR3L in the tumor-bearing mice led to the upregulation of MHCII and costimulatory molecules (e.g., CD80 and CD86), lowered expression of inhibitory molecules (e.g., TIM3), and significant tumor regression in an IFN-αβ signalingdependent manner [108].

Myeloid-derived suppressor cells (MDSCs)
MDSCs which are defined as immature myeloid cells with immunosuppressive properties are classified into two subtypes based on morphology and cell surface markers: monocytic MDSCs (M-MDSCs) and polymorphonuclear/ granulocytic MDSCs (PMN-MDSCs) [109]. They contribute to generating a tumor immunosuppressive milieu by induction of regulatory T cells and producing the arginase (ARG1), inducible NOS (iNOS), and inhibitory cytokines such as IL-10 and TGF-β [109]. As described by Marvel et al., elimination of these cells with low-dose chemotherapy or targeting their suppressive activity via various strategies such as using synthetic triterpenoid, phosphodiesterase-5 (PDE-5) inhibitor, cyclooxygenase-2 (COX-2) inhibition, and nitroaspirin, respectively, reduced the production of reactive oxygen species (ROS), arginase (Arg), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), and NO production and mitigated the immunosuppressive environment of tumors [109]. Thereby, the functionality of the infiltrated CAR-modified T cells is affected by the presence of MDSCs in the tumor microenvironment. The research conducted by Burga and coworkers revealed that simultaneous administration of anti-Gr-1, anti-GM-CSF, and anti-PD-L1 antibodies (to suppress the MDSCs) with anti-CEA CAR-T cells improved the anticancer activity of transferred T cells [110]. Moreover, co-administration of poly I:C and alltrans retinoic acid (ATRA) with adoptive T-cell therapy (ATC) enhanced the effectivity of tumor-specific CAR T cells against tumor-bearing mice through attenuating the immunosuppressive potency of MDSCs [111,112]. On the other hand, overexpression of CD33 surface marker on MDSCs with an immunosuppressive feature in whole blood and tumor stroma has been proposed for MDSCs depletion by Gemtuzumab ozogamicin immunotoxin that subsequently reestablished T cell proliferation and also reactivated anti-GD2 CAR T cells, anti-mesothelin CAR T cells, and anti-EGFRvIII CAR T cells [113]. Similarly, NKG2D ligand expression on MDSCs increased the susceptibility of these cells to killing by NKG2D.Ztransduced NK cells and further augmented GD2-CAR T cell trafficking and cytotoxicity against tumor in neuroblastoma xenograft model containing MDSCs [114]. It is worth noting that some chemotherapeutic drugs, such as sunitinib, modulate the components of the tumor microenvironment (such as Treg and MDSCs). In a study conducted by Li et al., the combination therapy with sunitinib increased infiltration and proliferation of carbonic anhydrase IX (CAIX) targeting CAR T cells by reducing the frequency of MDSCs and upregulating the expression of the target antigen [115].
Considering all of the evidence presented in this section, it seems that depleting inhibitory cells present in the TME such as Tregs, TAMs, and MDSCs or establishing exhaustion-resistant CAR T cells could improve therapy success.

Expression of immune checkpoint molecules and immunosuppressive mediators
Chronic antigen stimulation of infiltrated CAR T cells in tumor sites leads to upregulated expression of inhibitory receptors (e.g., PD-1) and exhaustion of T cells in PDL1-dependent manner [116,117]. According to an investigation performed by John and colleagues, the upregulation of the PD-1 receptor corroborated with the following antigen-specific stimulation of anti-HER2 CAR-transduced T cells in co-culture conditions with HER2-positive cancer cell lines, especially in CD8 + T subtypes [118]. In this context, various studies have implied that blocking PD-1: PDL1 interaction with mAbs led to the rescue of exhausted CAR T cells in solid tumors (Fig. 2a) [119][120][121][122]. However, several factors restrict the application of this approach, such as the requirement for multiple administrations because of the short half-life of antibodies and immune-related adverse events (irAEs) [120,123]. In order to alleviate systemic toxicity, several groups localized the immune checkpoint inhibition into the tumor site using modified CAR T cells secreting PD-1 blocking scFv [123,124]. Production of anti-human PD1blocking scFv by CD19 or MUC16 ecto -modified CAR T cells augmented their killing activity against PDL1 + tumor cells [123]. Similarly, Suarez et al. modified anticarbonic anhydrase IX (CAIX)-targeted CAR T cells to secrete anti-PD-L1 antibodies that subsequently reversed T cells exhaustion and restored their cytotoxic activity in clear cell renal cell carcinoma (ccRCC) [125].
THZ1 is an epigenetic modulator that selectively impedes CDK7 and alleviates the immune resistance induced by the expression of immunosuppressive genes (such as PDL1) [126]. As elucidated by Xia et al., combination therapy with EGFR targeting CAR T cells and THZ1 diminished the immune resistance and potentiated the efficacy of infused CAR T cells in triple-negative breast cancer (TNBC) [127].
Another strategy is to genetically modify CAR T cells to express PD1 dominant-negative receptors (DNR) or PD1: CD28 switch receptors (by exchanging transmembrane and intracellular domain of PD1 with CD28) that interfere with PD1 inhibitory signaling and thus resistant to the PDL1 overexpression in TME [116,[128][129][130][131][132]. On the other hand, employing the CRISPR-Cas9 gene-editing system to disrupt the expression of immune checkpoint molecules in CAR T cells (e.g., PD1, CTLA4, and LAG3) makes them more efficient (Fig. 2b) [133][134][135]. In this regard, Rupp and colleagues utilized Cas9 RNP to ablate PD1 expression and generate PD1 knockout anti-CD19targeted CAR T cells [136]. The selected number of clinical trials that used CAR T cells in combination with PD1 blockade against solid tumors is listed in Table 1 (from clinicalTrials.gov).
In addition to immune checkpoint molecules, the presence of various inhibitory mediators generated by the cancer cells itself or other tissue-resident cells impair the efficacy of infused T cells. For instance, indoleamine 2,3-dioxygenase (IDO) is involved in the conversion of the tryptophan to immunosuppressive metabolites (i.e., kynurenine and 3-hydroxyanthranilic acid (3-HAA)) which hinders T cell's proliferation and effector function [137]. As noted by Ninomiya et al., preconditioning lymphodepleting chemotherapy before anti-CD19 CAR T infusion (e.g., fludarabine and cyclophosphamide administration) altered tumor immunosuppressive milieu by suppressing IDO expression, which in turn, enhanced the activity of infused cells against IDO positive cancer cells [137].

Tumor heterogeneity
Solid tumors are heterogeneous on multiple levels, comprising heterogeneity between patients (inter-tumoral heterogeneity), differences between tumors in the various site in an individual (inter-site heterogeneity), and differences among cell populations within a single tumor (intratumoral heterogeneity) [138][139][140]. Neoplasm heterogeneity between patients was leading to various clinical outcomes. Hence, genetic and cellular tumor heterogeneity is reflected in patient response to anticancer therapy and must be taken into consideration when employing targeted therapy at the large-scale level [141].
On the other hand, the heterogeneity has been documented in the tumor microenvironment or infiltrated immune cells during disease progression or at the primary and metastatic sites. Intratumoral heterogeneity that is common at the genetic mutation, gene expression, and protein post-translational modification levels is limiting the effectiveness of therapeutic approaches [142]. As noted by Horton et al., the heterogeneity and somatic mutation of tumor cells altered intrinsic signaling pathways contributed to the development of non-T cell-inflamed tumor microenvironment (i.e., tumors lacking T-cells) by affecting the antigen-presenting cells and T cell infiltration [143]. Accordingly, small molecules targeting the intrinsic signaling pathway of tumor cells could promote T cell accumulation.

Antigenic heterogeneity of tumor cells
Identifying the surface antigens of tumor cells (including proteins, carbohydrates, and glycolipids) by CAR molecules is essential for CAR T cell activation. Therefore, selecting a target with high coverage, specificity, and stability plays a fundamental role in the complete clearance of tumor cells and is more complicated in heterogeneous solid tumors [144]. Tumor cells escape from immune recognition by modulating the expression of the target antigens; hence, due to the high mutation rate and heterogeneity of the tumor antigens, one of the challenges facing the CAR T cell therapy approaches is specific and efficacious targeting [145,146]. In a study accomplished by Rourke et al., antigen loss or mutation of target antigens are common after CAR T cell infusion in solid tumors [22]. Applying the additional target is one possible solution recommended by the authors.
It has been verified that simultaneous targeting of multiple tumor antigens by CAR T cells or targeting tumorsupportive cells has superior anti-tumor efficacy [144]. Indeed, simultaneous targeting of at least two antigens on the surface of cancer cells reduces the probability of tumor escape via downregulation/loss of the antigen expression. In this context, several studies created a tandem-CAR (TanCAR) molecule that enabled the T cells to recognize two distinct target antigens. These transduced T cells stimulated with either target or simultaneous recognition of both targets enhanced cell performance [147,148]. Moreover, combinatorial administration of two different redirected CAR T cells such as anti-PSCA-and anti-MUC1-specific CAR T cells (in 1: 1 ratio) in the non-small cell lung cancer (NSCLC) PDX model was Table 1 Selected clinical trials using PD-1 disrupted CAR T cells against solid tumors (from clinicalTrials.gov) more efficacious than each of them, according to Wei et al. [149]. However, many studies have demonstrated that dual CAR-expressing T cells, which expressed two distinct receptors, have superior anticancer activity than pooled CAR T cells, composed of the T cells with different receptors [150,151]. Furthermore, as demonstrated by Anurathapan et al., incorporating the epigenetic modulators such as decitabine as a hypomethylating agent in combination with CAR T cells increased the expression of tumor-associated antigens (e.g., MUC1), thus sensitizing cancer cells to be recognized and killed by T cells [146]. Overall, it would be worthwhile to screening patients for target antigen expression and selecting them if the percentage of expressing cells exceeds a predetermined level [152].

Normal tissue toxicity
Another restriction factor in applying genetically engineered T cells in solid tumors is their on-target/offtumor toxicity [153]. Due to the limitation in access to truly tumor-restricted antigens, the tumor-associated antigens (which have low expression in healthy tissue) are usually employed for tumor targeting by CAR T cells, which cause TAA-targeted T cells toxicity against healthy tissue. For instance, liver toxicity observed following anti-CIAX-redirected T cells infusion in patients with metastatic renal cell carcinoma (RCC) as well as inflammatory autoimmune colitis revealed after treatment with carcinoembryonic antigen (CEA)-specific TCR-engineered T cells in patients with metastatic colorectal cancer [153,154]. Likewise, as reported by Morgan et al., low-level expression of ERBB2 on lung epithelial cells caused T cell accumulation in the lung immediately after intravenous injection of anti-ERBB2-targeted CAR T cells which led to cytokine storm, respiratory distress, and death [155].
Transduction of T cells with lower affinity CARs restricted their optimal activity to tumor cells with overexpressed target antigens, thus reduced their cytotoxic effects against normal tissue expressing lower levels of target antigens [156,157]. On the other hand, dissociation of the signaling domains of CARs (which includes activation and costimulation signals) in two different antigen-specific receptors and subsequent trans expression of both CARs on T cells reduced the CAR T cells response against normal cells expressing only one target [158]. As recommend by Kloss and coworkers, it is preferable to use the chimeric costimulatory receptor (CCR) (i.e., engineered CAR specific for the second antigen contains the costimulatory domain of CD28 and 4-1BB molecules) for targeting the antigen that is expressed on normal tissue and has a more deleterious side effect [159]. It should be noted that only dual antigens expressing cells are recognized by these modified T cells, and thereby tumor cells expressing one target antigen are disregarded.
According to Roybal et al., CAR T cells generation using Boolean AND logic gate strategy confined the CAR expression of T cells to the detection of localized tumor microenvironment antigens, thus specify CAR T cell activity against multiple antigens expressed in TME [160]. Similarly, Wu and colleagues applied small molecules for assembling antigen-binding compartments and intracellular signaling events [161]. In this situation, the concentration and timing of Rapalog addition (modified rapamycin) controlled the T cell activation when exposed to the cognate antigen (CD19) [161]. Especially, tacrolimus includes the same binding site in FKBP as rapamycin analog, so it modulated CAR surface detection by competing with rapamycin and could be exploited as an additional regulator for CAR T cell activity [162].
More than CAR signaling, which can be controlled by small molecules, switching-on systems, such as the doxinducible Tet-On system, have been used to regulate the expression of these receptors. Accordingly, integrating the tetracycline-on system in the CAR construct modulated CAR expression in the presence of doxycycline and served as a remote control for CAR T cell activation after infusion into a patient [163]. Similarly, restricting the CAR expression to hypoxia condition of TME using an oxygen-sensing system mitigated the on-target/off-tumor toxicity of ERbB-targeted CAR T cells [164]. Restriction of CAR expression via electroporation of mRNA or administration of monoclonal antibodies to shield the target antigens expressed on the surface of normal cells are two strategies recommended to minimize CAR T cell off-target toxicity [165,166].
The evidence reviewed here suggests a pertinent role for managing the tumor microenvironment to leverage the CAR T cell approach. Table 2 highlights various strategies used for improvement of CAR T cell therapy in clinical trials (from clinicalTrials.gov).

CAR-T cell's next generations
Despite its widespread success in cancer treatment, particularly in hematological malignancies, CAR T cells are a personalized and expensive product that has become out of reach for most patients. Therefore, genetic engineering of T cells from a healthy donor instead of patient T cells saves time and cost. The inspiring clinical results of CAR-T cell therapy can potently be more intensified through the generation of the more potent and histocompatible T cells. While T cells can be simply obtained from donors, their employment is mainly bargained because of the high alloreactive competence [167] and eventually results in the graft rejection in transplant recipients as well as GVHD in recipients of donor-procured T cells [168]. It seems that dual signaling transduction of TCR and CAR may be involved in GVHD occurrence through the induction of exhaustion [169], chronic activation, and activation-induced cell death (AICD) of allogeneic CAR T cells [170]. Besides, HLA expression on allogenic-CAR T cells stimulates recipient immune response and limits the persistence of infused cells. By this fact, it is beneficial to disrupt endogenous TCR signaling and diminish   HLA expression on allogeneic CAR T cells to reduce GVHD and evade alloreactive T cell attack [170,171]. These modified cells, known as universal CART cells, can be given to any patient irrespective of their HLA status. Growing evidence has currently indicated that three main genome-editing tools, containing zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more potently clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 can provide the opportunities to gene disruption in the CAR T cell (Table 3) [171,172]. Notably, these methods enhance transduction efficiency, reduce GVHD, and improve the infused T cell persistence in cancer patients. Interestingly, the knock-out of endogenous TCR expression using genome-editing tools can support manufacture of universal CAR-T cells [173,174]. In fact, universal "off-the-shelf, " or allogeneic, CAR T cells is an alternative that can efficiently defeat such issues and support the Table 3 Summary of preclinical reports based on manufacture of next generations CAR T cells using genome editing technologies   [188,189] In vivo

Leukemia
In vitro TRAC TALEN CD20 [195] PD-1 various modifications and CAR combinations to affect various tumor antigens and avert tumor evasion. In addition to these valued attributes, probable side effects of universal CAR T cells could be compromised by a customization of the adaptor dose [175].
Recent studies have shown that switchable universal CAR-T cell to target CD123 eliminated CD123 + leukemia in vitro and also these cell bearing murine in vivo. As well, CD123-redirected universal CAR-T exhibited reversible toxicity versus hematopoietic cells than normal CD123-CAR-T cell [176]. Moreover, the CRISPR-Cas9 mediated ablation of TCR, beta-2 microglobulin (B2M) and PD-1 concurrently leads to the preparing the CAR-T cells with higher anti-tumor activities than conventional CAR-T cells. Further, TCR and HLA class I double deficient T cells usually display reduced alloreactivity and universally result in no GVHD occurrence [133,134]. Besides, PD-1-deficient EGFRvIII-directed CAR-T cells could elicit robust inhibitory effects in on EGFRvIII positive glioblastoma cells in vitro [177]. Similarly, lymphocyte activation gene-3 (LAG-3) deficient CD19-directed CAR-T cells could exert evident antigen-specific antitumor impacts in vitro and in vivo against leukemic cells [135]. On the other hand, GM-CSF knocked-out CD19specific CAR-T cells proficiently could produce lower levels of GM-CSF, leading to the less cytokine releases storm syndrome (CRS) [178]. In another reports, TGF-β receptor II (TGFBR2) ablation by genome-editing technologies upgraded anti-tumor efficacy of anti-mesothelin CAR-T cells against ovarian cancer cells, which mainly mediated by a reduction in the activated Treg conversion and circumventing CAR-T cells depletion [179]. Also, TGFβRII-deficient CAR-T cells could exhibit remarkable resistance to the TGFβ inhibition and thereby induce extended cytotoxicity against tumor cells [180]. A summary of studies in this regard are cited in Table 3.

Conclusion
Following CAR-modified T cells striking success in hematological malignancies, they have been proposed as a novel curative strategy in solid tumors. The application of engineered cells specific for TAA antigens such as mesothelin, CEA, GD2, MUC-1, EphA2, and HER2 exhibited promising outcomes in solid tumors. However, it has not been as effective as hematological malignancies and has encountered challenges such as lymphocyte penetration into the tumor tissue or exhaustion by the immunosuppressive tumor milieu [181].
One of the drawbacks of CAR T cell therapy is CRS, which is proportional to the tumor burden. Because overactivation of the immune system and elevated serum levels of inflammatory cytokines, i.e., IL-6 and IL-1, are involved in the pathophysiology of CRS, systemic administration of corticosteroids, blockade of the IL-6 receptor, and generation of engineered CAR T cells that secret IL-1 receptor antagonists have all been proposed as ways to manage the side effects [182]. As noted by Ng and coworkers, incorporating the cytoplasmic domain of DAP12 with only one immunoreceptor tyrosine-based activation motif (ITAM) into the CAR construct mitigated the level of cytokine secreted from stimulated CAR T cells as well as the occurrence of CRS [183].
As indicated earlier, the presence of regulatory cells, overexpression of immune checkpoint molecules, and hypoxia condition of TME exhaust the adoptively transferred T cells and impair their proper anti-tumor activity. On the other hand, the specific features of malignant tissue such as tumor vasculature and the extracellular matrix hamper CAR-T cell penetration. Accordingly, several groups have exploited various strategies to surmount these challenges, such as combination therapy (e.g., immune checkpoint blockade mAbs), depletion of regulatory cells, generation of resistance cells, and armored CAR-T cells. Besides, cyclophosphamide preconditioning enhanced the efficacy of the CAR T cell therapy approach by impeding the immunosuppressive TME and fostering a proinflammatory milieu [184]. In the study conducted by Cha and coworkers, anti-CEA-IL-2 immunocytokine treatment (CEA-specific antibodies fused to IL-2) after cyclophosphamide plus CEA-specific CAR T cell administration enhances CAR T cell cytotoxic activity [185]. Similarly, treatment with a low dose of docetaxel modifies the tumor microenvironment and improves PSMAspecific CAR T cell infiltration into the tumor bed, according to Alzubi et al. reports [186]. Overall, identifying the inhibitory mechanisms and employing counteracting approaches improve the function of endowed cells in solid tumors.