CAR T cell therapy as a promising approach in cancer immunotherapy: challenges and opportunities

doi:10.21203/rs.3.rs-203368/v1

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CAR T cell therapy as a promising approach in cancer immunotherapy: challenges and opportunities

https://doi.org/10.21203/rs.3.rs-203368/v1

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published 24 Mar, 2021

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Background Chimeric antigen receptor (CAR)-modified T cell therapy has shown great potential in the immunotherapy of patients with hematologic malignancies. In spite of this striking achievement, there are still major challenges to overcome in CAR T cell therapy of solid tumors, including treatment-related toxicity and specificity. Also, other obstacles may be encountered in tackling solid tumors, such as their immunosuppressive microenvironment, the heterogeneous expression of cell surface markers, and the cumbersome arrival of T cells at the tumor site. Although several strategies have been developed to overcome these challenges, aditional research aimed at enhancing its efficacy with minimum side effects, the design of precise yet simplified work flows and the possibility to scale-up production with reduced costs and related risks is still warranted.

Conclusions Here, we review main strategies to establish a balance between the toxicity and activity of CAR T cells in order to enhance their specificity and surpass immunosuppression.

In recent years, many clinical studies have been conducted that eventually led to approved products. To date, the FDA has approved two anti-CD19 CAR T cell products for non-Hodgkin lymphoma therapy, i.e., axicbtagene ciloleucel and tisagenlecleucel. With all the advances that have been made in the field of CAR T cell therapy for hematologic malignancies therapy, ongoing studies are focused on optimizing its efficacy and specificity, as well as reducing the side effects. Also, the efforts are poised to broaden CAR T cell therapeutics for other cancers, especially solid tumors.

Oncology

Cancer Biology

CAR T cell therapy

Cancer

Immunotherapy

Adoptive cell transfer

Cancer immunotherapy is referred to as any form of intervention that forces the immune system to eliminate malignancy. Successful immunotherapy produces anti-cancer immune responses, which are systemic, highly tolerable and overcome the limitations of traditional treatment approaches [1]. Adoptive cell transfer (ACT) is regarded as a novel and efficient immunotherapeutic strategy, especially for cancer treatment [2]. This approach involves the isolation and in vitro expansion of a cancer patient-deriveds activated T lymphocytes and their subsequent reinfusion into cancer patients along with appropriate growth factors to stimulate their in vivo survival and proliferation [2]. It has been shown that the reinfusion of tumor-infiltrating lymphocytes (TILs) may result in cancer regression, but obtaining high-affinity T cells can be complicated by an immunosuppressive tumor microenvironment. Also, since the accessibility of TILs may be accompanied by technical limitations in at least some tumors, TIL reinfusion is not considered to be fully effective for all tumors [3, 4]. Hence, modification of T cells towards a highly activated form can be an alternative strategy to efficiently combat various forms of cancers. In recent years, chimeric antigen receptor T cells (CAR T cells) have been introduced to efficiently recognize cancer antigens and to accurately kill tumor cells [1, 5].

Chimeric antigen receptors (CARs) are receptor proteins that have been engineered to provide T cells with the ability to target specific proteins. In CAR T cells, the antigen recognition moieties of the engineered receptors are derived from monoclonal antibodies (mAbs). Thus, in addition to the cytolytic efficacy of T cells, CAR structures endow T cells with the enormous diversity and high-affinity/specificity of mAbs against tumor antigens. In addition, CARs can tackle a broad range of candidates for targeting in a major histocompatibility complex (MHC)-independent fashion. In contrast to TILs, this property enables CAR T cells to overcome MHC expression down-regulation by tumor cells (immune escape) [6–8]. In preclinical and clinical CAR T cell studies, various CAR designs are being assessed. These designs mainly involve the antigen-binding domain, the intracellular domain, the transmembrane domain and its spacer (Fig. 1a).

2.1 Antigen-binding domain

The extracellular CAR domain recognizes target antigens on tumor cell surfaces and triggers CAR T cell attack. This CAR domain is connected to the transmembrane and intracellular domains by a hinge [9]. In most clinical and preclinical studies, single-chain variable fragments (scFvs) are used in CAR designs as antigen-binding domains. ScFv modules are composed of the reformed variable region of mAbs to the single-chain of heavy and light variable fragments that are connected together in tandem by a flexible linker [9]. In addition to the simple integration of different scFvs in CAR constructs, the use of scFvs as antigen-binding domains enables the targeting of a wide spectrum of antigens with high affinity and specificity. In contrast to traditional T cell receptors, scFvs also enable the targeting of soluble antigens. Therefore, scFvs are beneficial in targeting soluble immunosuppressive molecules present in solid tumor niches, such as transforming growth factor-beta (TGF-\({\beta })\)[10]. On the other hand, the application of scFvs in CAR constructs is complicated by a tendency for oligomerization, which may lead to tonic signaling and T cell exhaustion [11]. Most scFvs that are used in CAR designs are derived from murine models that are potentially reactive towards human anti-mouse antibodies [7]. Replacing human scFvs by their murine forms helps to prevent these effects.

Instead of scFvs, in various studies alternative binding domains have been used for specific functions, such as cytokines [12, 13], peptides [14], Fc receptors [15] and nanobodies [16]. Nanobodies, for instance, are single-domain antibodies that are derived from camelid, comprising the variable regions of heavy-chain antibodies, leading to higher resistance and lower immunity. In contrast to scFv-based CAR T cells, nanobody-based CAR T cells suffer from fewer encounters with conformational deficiencies. Moreover, their small sizes endows CAR T cells with proper characteristics, thus efficiently infiltrating rigid solid tumor extracellular microenvironments [17, 18].

2.2 Intracellular domain

The intracellular domain of CARs is commonly composed of two components [19]: (i) a cluster of differentiation 3 (CD3) ζ chain, which is a necessary component of the intracellular domain and consists of immunoreceptor tyrosine-based activation motifs (ITAMs); these motifs are derived from T cell receptor (TCR) co-receptors and play important roles in signal transduction [20], and (ii) one or more costimulatory domains, which help the complete activation of CAR T cells and their persistence. Interestingly, the selection of different costimulatory domains is one of the most effective factors determining CAR T cell functioning, metabolism and phenotype [21].

CD28 and CD4-1BB are the most common costimulatory domains that are used in CAR designs and exhibit dramatic anti-tumor efficacies. CD28 CAR T cells show an effector-like T cell phenotype and a potent cytolytic function. Nevertheless, these cells exhibit a short half-life and a low expansion rate in vivo [22, 23] and are more prone to T cell exhaustion [22]. In contrast, modified T cells with CARs consisting of CD4-1BB emerge as memory-like T cells, which exhibit a high expansion rate and persistence while showing less action severity in vivo [23, 24]. The different behaviors of CD28 and CD4-1BB CAR T cells are attributed to disparate downstream signaling pathways that are triggered by the subsequent activation of T cells [22]. Despite these different functions, CD28 and CD4-1BB CAR T cells do not exhibit significant differences in anti-tumor activities or remission rates in clinical trials [25]. Notably, CD28 and CD4-1BB are not the only costimulatory domains that are used in CAR designs. Several preclinical studies have used other endodomains as costimulatory domains, such as myeloid differentiation primary response 88 (MYD88)/CD40 [26, 27], CD27 [28], CD134 [29, 30], inducible T cell costimulator (ICOS) [31, 32] and DNAX activating protein 12 (DAP12) [33]. As yet, however these alternative costimulatory domains have not been evaluated in patients and, therefore, the efficiencies of these different domains still need to be determined precisely.

2.3 Transmembrane domain and spacer

The transmembrane domain normally consists of a hydrophobic alpha-helix, which is usually derived from type I proteins, such as CD3ζ, CD28, CD4 or CD8α [34]. This domain tethers CARs to the cell membrane and connects the antigen-binding site to the intracellular domain and is, therefore, fundamental for CAR surface expression and stability [35, 36]. The transmembrane components may affect CAR stability, functioning and downstream signaling. CD28 has, for instance, a greater impact on CAR stability than CD3ζ [37]. Also, CD3ζ may aid CAR-mediated T cell activation due to its role in CAR dimerization and incorporation into endogenous TCRs [35].

In some cases, recognition domains are linked to transmembrane domains by a flexible short peptide fragment as a spacer that is usually derived from CD8, CD28, immunoglobulin G (IgG)1 or IgG4 [34]. Optimal lengths of the spacer provide a balance between the proximity of scFvs and cognate epitopes. They also play an important role in the conformational freedom of CAR receptors for successful attachment [38–40].

Most CAR T cells used in clinical trials can be divided into four generations (Fig. 1b). The first generation of CARs is composed of an extracellular domain along with an intracellular domain, which only consists of CD3ζ. This intracellular domain mediates the signaling cascades after antigen recognition and activation. Since the first generation of CAR T cells encountered deficiencies in their expansion and activity in vivo [11, 41, 42], second and third generation CAR T cells were constructed by adding one or two costimulatory domains, respectively [43–45]. To increase the efficiency of CAR T cells, a fourth generation of CAR T cells, also known as CAR T cell redirected for universal cytokine killing (TRUCK), was developed [18, 36]. In this optimized design, after antigen recognition, CAR T cell activation is followed by cytokine and chemokine induction, leading to self-additional activation. The fourth generation of CAR T cells enables the recruitment and activation of endogenous immune cells and, therefore, their dominance over the immunosuppressive tumor microenvironment (TME) [46]. These CAR T cells were additionally engineered with a nuclear factor of activated T cell (NFAT)-responsive expression cassette (in different genes in the same vector) for the inducible expression of a transgenic cytokine [47]. To date, several cytokines have been adopted into TRUCK designs, including interleukin 12 (IL-12) [48], IL-18 [49] and TNF receptor superfamily member 14 (TNFRSF14).

Despite the advantageous opportunities of CAR T cell therapy, especially in hematological malignancies, several obstacles still limit the general application of this therapeutic strategy. Traditional drug-based cancer therapy depends on general target approaches, while CAR T cell therapy relies on the distinction between cancerous and healthy tissues, as well as on active targeting. However, by not fully reaching this goal, CAR T cell therapy has faced a complex challenge of off-target effects [50] and off-target related toxicity. In addition, true tumor-targeting can lead to CAR T cell over-activation, highly increased cytokine secretion and severe general toxicity. In addition, tumor immune escape has been reported in a considerable number of CAR T cell therapies, and many studies have addressed immune escape with different strategies based on multi-target approaches.

A major challenge in CAR T cell therapy is encountered in patients harboring solid tumors. This challenge can be attributed to several factors, such as lack of suitable tumor-specific antigens, immune-suppressive properties of the TME, tumor heterogeneity, and CAR T cell migration and homing. CAR modifications capable of overcoming these obstacles may extend the therapeutic applications of CAR T cells. Here, some of the challenges and the main implemented strategies in overcoming these obstacles are discussed.

4.1 Toxicity

As mentioned above, toxicity is a challenging issue that can be categorized into two clusters: (i) general toxicity as an over-activation-based toxicity and (ii) off-target-based toxicity. General toxicity arises from cytokine secretion at a toxic level, following a robust interaction of CAR T cells and cancer cells. In some cases, intense interaction between CAR T cells and host immune cells results in cross-activation and a cytokine storm that causes toxicity. General toxicity syndromes that occur after CAR T cell therapy include cytokine release syndrome (CRS) [51], immune effector cell-associated neurotoxicity syndrome (ICANS) [52] and, in rare cases, hemophagocytic lymphohistiocytosis (HLH) [53] and macrophage activation syndrome (MAS) [53].

CRS or a cytokine storm is a drastic toxic situation that is frequently observed in patients who have received CAR T cell therapy [15]. This complication is ascribed to expansion and over-activation of CAR T cells, accompanied by a high secretion of various cytokines, such as IL-6, IL-10, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) or granulocyte-macrophage colony-stimulating factor (GM-CSF) by CAR T cells and other immune cells [54]. The severity and kinetics of CRS are influenced by several factors, including overall tumor burden, antigen density, antigen-binding domain affinity and costimulatory factors. For instance, CARs containing CD28 costimulatory domains are, due to their intensive functions, associated with a higher CRS risk than those containing CD4-1BB. Hence, it seems that a selection of costimulatory domains must be performed while regarding other risk factors such as tumor burden and antigen density.

CRS symptoms reported in clinical trials have been classified into five grades, ranging from mild to severe toxicity [55]. Fever, myalgia, fatigue and mild hypotension are considered mild CRS symptoms and are manageable by supportive care and by diminishing side effects such as pain. Conversely, hypotension requiring vasopressors, respiratory failure, coagulopathy and multi-organ system failure are considered severe complications of life-threatening and lethal grades of CRS [55]. In order to reduce severe levels of CRS, several strategies have been proposed: (i) sequential cytokine level assessment that can be useful for early detection and inhibition of toxicity progression, (ii) multiple CAR T cell reinfusion with increasing doses, which is advised to control CRS, (iii) use of immunosuppressive drugs such as Tocilizumab (anti-IL-6 receptor), siltuximab (anti-IL-6 antibody) or corticosteroids, administered for CRS suppression and treatment; it must be noted, however, that immunosuppressive drugs can also reduce the functionality of CAR T cells [56] and, therefore, the use of other agents such as IL-1β inhibitors [57], TNF-α inhibitors [58] and GM-CSF inhibitors [59] may be of help to minimize CRS, although their exact efficacy is still elusive, and (iv) the construction of switchable CAR T cells in managing severe toxicity.

Neurotoxicity is another form of prominent toxicity that usually accompanies CRS. The exact mechanism of CAR T cell-associated neurotoxicity has not been elucidated yet [60]. Moreover, it seems that several mechanisms may contribute to the development of neurotoxicity, including passive or active leakage through the blood-brain barrier (BBB) and local accumulation of cytokines and immune cells in the central nervous system [61]. The most common symptoms of ICANS include headache, seizures, delirium, anxiety, tremor, aphasia, decreased consciousness and coma with cerebral edema. In general, mild clinical symptoms are transitory, reversible and self-improvable [62]. In severe situations, IL-6 pathway inhibitors and corticosteroids can be useful in managing neurotoxicity during both the onset of symptoms and at late stages [63]. In more stressful conditions, patients suffering from epilepticus with convulsive or no convulsive status should be treated with benzodiazepines and additional anti-epileptic drugs [63].

4.2 Immune escape

In a considerable number of CAR T cell therapies, such as CD19 CAR T cell therapy, fully or partial loss of target antigens on cancer cell surfaces has been reported [64]. This phenomenon can be explained by several mechanisms. One is CD19 loss on the tumor cell surface due to a mutation that leads to alternative splice variants in exon 2 of the CD19 gene, which appears to reduce CD19 cell surface expression [65]. Other studies showed that splice variants in exons 5 and 6 of the CD19 gene lead to a truncated form of CD19 that prevents binding to the target antigen [65, 66]. There exists conflicting information on to whether splice variants are already present [67] or arise as a result of CAR T cell therapy [68]. The presence of CD19 isoforms in some patients with B-cell acute lymphocytic leukemia (B-ALL) suggests another mechanism for antigenic escape [68], i.e., lineage switching from a CD19 + B-ALL tumor cell phenotype to CD19 myeloid cells [69]. In rare cases [70], inaccurate isolation of T cells during the process of CAR T cell production has been found to result in the infusion of CAR molecules into leukemic B cell blasts. During the process of CAR T cell therapy, the expressed CD19 markers on leukemic B cells are masked by their own adjacent anti-CD19 CAR, thus preventing anti-CD 19 CAR T cell attack. Various studies have addressed tumor immune escape using a multi-target approach, some of which will be discussed below.

4.3 CAR T cell specificity

Another challenge in CAR T cell immunotherapy is associated with ‘on-target, off-tumor’ complications (Fig. 3A). In other words, some target antigens do not exhibit an exclusive expression on tumor cells but, instead, exhibit varied levels of expression on normal tissue cells as well [71]. As a result, besides tumor targeting, CAR T cells may also target healthy tissues, leading to adverse effects on vital organs. This complication has also been observed in anti-CD19 CAR T cell therapy, which is approved by the US food and drug administration (FDA). CD19 is also expressed in normal B cells [72]. Therefore, following CD19 CAR T cell therapy, normal CD19 + B cells are eradicated, leading to hypogammaglobulinemia. This side effect is manageable with sequential intravenous infusions of immunoglobulin, replacing the eliminated B cell-derived antibodies [72]. In contrast, in other types of cancer, especially solid tumors, ‘on-target off-tumor’ targeting may cause more acute problems and be less tolerable. Off- target-based toxicity has been reported in some CAR T cell therapies against solid tumors, such as CAR T cell therapies targeting human epidermal growth factor receptor 2 (HER2), carbonic anhydrases IX (CAIX) and mesothelin. This issue limits the clinical potential of many CAR T cell products. Therefore, several strategies are being adopted to generate more efficient clinical CAR T cell products that include detecting and implementing antigens with higher specificity to tumor cells, optimizing the interaction of CARs with tumor cells in comparison to healthy cells, and multi-targeting of different tumor-specific antigens that increases the possibility of accurate targeting.

As mentioned above, it appears that CAR-T cell-related toxicity and specificity are the most important challenges that should be addressed. Therefore, several approaches are taken to overcome these issues. Some strategies are being implemented to efficiently control and manage CAR T cell activity and avoid adverse toxicity. Other studies focus on the prevention of CAR T cell activation by undesired targets. Several critical strategies, developed for safety and specificity improvement of CAR T cell therapy, are dealt with in this review.

Excessive cytokine secretion by CAR T cells limits the clinical efficiency of CAR T cell therapy and increases the possibility of life-threatening side effects. Thus, the discovery of methods that provide opportunities to control the initiation, termination and CAR T cell activity is warranted. To this end, several methods have been developed to manage CAR T cell activity via silencing or eliminating CAR T cells after increased toxicity (Fig. 2).

5.1 Suicide switchable CAR T cells

An approach based on a suicide gene represents an off-switch strategy that is suggested to control systems and reduce excessive toxicity of CAR T cells. For this purpose, inducible safety switch segments are integrated into CAR constructs so that chemical factor administration can induce CAR T cell death and elimination. For instance, CAR T cells that are transduced with inducible caspase 9 (iCasp9) safety switches can mediate conditional apoptosis upon treatment with a chemical inducer of dimerization (CID) [73] (Fig. 2A). This strategy may increase the safety of CAR T cell therapy. However, immediate apoptosis induction in most of the modified CAR T cells results in an irreversible termination of CAR T cell therapy and, thus, disease progression [74]. Alternative suicide genes are epitope tags that are recognized by FDA-approved mAbs and induce T cell death through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In this approach, programmed CAR T cells express CD20 or truncated epidermal growth factor receptors (EGFRt). The application of associated mAbs, rituximab for CD20 or cetuximab for EGFRt, results in efficient and specific elimination of CAR T cells. Possible on-target side effects arising from mAb binding to normal tissue cells limit further development.

5.2 Induced functional CAR T cells

Other strategies may also positively regulate CAR T cell activity. In this case, CAR T cells are activated using exogenous user-provided signals, such as small molecules. The insertion of an inducible gene regulatory system may, for instance, enable controlled expression of CARs upon drug administration, such as tetracycline and doxycycline [75] (Fig. 2C). In this regard, a Tet-On inducible system has been used to control CD19 CAR T cell activity, with a significant antitumor effect in xenograft models [75]. This system allows the reversible control of CAR T cells. However, the relatively slow induction procedure, the partial expression of CARs and some background CAR expression in the absence of inducer molecules may limit the efficacy of this approach. In another strategy, through an induced assembly approach, functional CAR T cells are formed [76]. In this approach, CAR receptors split into two non-functional fragments, one fragment containing the antigen-binding domain and the other containing the activation domain. Each fragment possesses an inducible assembly domain with a binding tendency to the same small molecule. Furthermore, the addition of cognate small molecules assembles the split receptors and provides functional CAR receptors (Fig. 2B) [76]. The limited life span of small molecules restricts prolonged CAR T cell activity in a small molecule-dependent manner, whereas the sequential application of small molecules enables CAR T cell activity.

5.3 Zip CAR T cells

Recently, split universal and programmable CARs, which are also known as SUPRA CARs, have been designed to regulate CAR activity. In this system, the CAR segment consists of two moieties: zipCAR and zipFv. Each moiety includes a leucine zipper segment that is attached to a signaling domain in the zipCAR section and to a ligand-binding scFv domain in the zipFv segment. Vacant zipCAR and zipFv moieties with matched leucine zipper segments are attached, forming a functional CAR T cell (Fig. 2D) [77]. One of the benefits of SUPRA CAR T cells is the capability of controlling CAR T cell activity through external zipFvs. Adding external zipFvs with a complementary structure to the leucine zipper of the original zipFv creates a dimer with zipFv and, thus, inhibits the formation of a functional CAR and its activity. Alternatively, compared to the desired zipFv, the use of external zipFvs at a high density or tendency to the zipCAR segment can lead to domination of a regulatory zipFv over its original, thereby terminating CAR T cell activity (Fig. 2D). This strategy has not only been applied to a safety switch to manage CAR T cell activity, but is also used for either sequential or simultaneous multi-targeting. This approach provides the opportunity to target a panel of antibodies with the same engineered T cells [78]. Different zipFvs can be used without re-administration of CAR T cells, based on the patient's responses during treatment. This method may also be effective for highly heterogeneous solid tumors because of its multi-target capacity [77].

5.4 Multiple antigen recognition

Multiple targeting is an effective strategy that allows CAR T cells to discriminate normal cells from tumor cells, thus targeting true tumor cells. This strategy can also be used for surmounting the immune escape of tumor cells. The sequential infusions of different CAR T cells directed against different antigens may be clinically effective. Multi-target CAR T cell therapies can be created by transducing T cells with different CAR constructs, harboring different scFv sequences (Fig. 3B). Also, engineering strategies that produce a single CAR with different targets are being considered.

5.5 SynNotch CAR T cells

Synthetic Notch (synNotch), a form of “AND logic gate” system, increases the specificity of CAR T cells by engaging multiple tumor antigens that are needed for full CAR T cell activation. In this system, the binding of “A” antigen to synthetic Notch receptors leads to the release of a Notch transcription factor to the nucleus and the subsequent induction of CAR expression against the “B” antigen (Fig. 3E) [79, 80]. Therefore, SynNotch CAR T cells are activated in response to a simultaneous recognition of A and B tumor antigens [79, 80]. In a recent study, engineered T cells with synthetic Notch (synNotch) receptors specific for epithelial cell adhesion molecule (EpCAM) or CD276 that induce receptor tyrosine kinase-like orphan receptor1 (ROR1) CAR expression, were applied to distinguish tumor cells expressing ROR1 from bone marrow stromal cells and splenic stromal cells, also expressing ROR1. The results showed an increase in tumor-cell specificity, reducing the risk of toxicities in animal models [81]. Although this system increases CAR T cell precision, the slow process of CAR T cell activation may limit its efficiency.

5.6 Split CAR T cells

In Split CAR T cells, the CAR segment is separated into two non-functional receptors. In each receptor, one antigen-binding domain is accompanied by a costimulatory domain and the other by a CD3ζ activation domain (Fig. 3D). In this design, the two receptors target different antigens. Therefore, the CAR T cell activity depends on the simultaneous recognition of both antigens [76]. Preclinical models of both breast [82] and prostate [83] cancer have shown promising results of this approach.

5.7 Tandem CAR T cells

Tandem CAR T cells, also known as “OR gates”, consist of two scFv domains that are tandemly oriented and work under the same intracellular signaling domain. These antigen-binding sites target different antigens (Fig. 3C). The attachment of each of the binding sites can activate CAR T cells. However, functional synergy and enhanced activation only occur when they are simultaneously engaged [84]. It has also been shown that tandem CAR T cells are more sustainable and less prone to T cell exhaustion and can dominate solid tumor TMEs [85]. Currently, several preclinical studies aimed at using tandem CAR T cells directed against CD19/CD20 and CD19/CD22 for B cell malignancies have been translated into phase 1 clinical trials.

5.8 CART.BiTE cells

The bi-specific T cell engagers (BiTEs)-expressing CAR T cells (CART.BiTE cells) approach is designed to tackle major obstacles in immune therapy, such as antigen escape and tumor heterogeneity. BiTEs are a class of engineered bispecific monoclonal antibodies that are formed from two scFvs, which are connected by a flexible linker. One of the scFvs binds to a particular tumor-associated antigen (TAA), whereas the other forms a specific bond with the invariant component of the TCR complex, CD3 [81]. The simultaneous attachment of BiTEs to CD3 and to a TAA generates an immune synapse between T cells and malignant cells, initiating the cytotoxic activity of T cells against these cells. A new approach combines CARs with BiTEs into a single gene-modified T cell product that co-expresses CAR and BiTE molecules. In this method, a bi-cistronic gene construct containing CAR and BiTE is transferred to T cells to generate CART.BiTEs, i.e., cells with surface CAR expression and BiTE secretion. CAR and BiTE in CART.BiTEs are designed against different antigens, and thus BiTEs secreted from CAR T cells can attach to the T cell receptor of the cell of origin and, thereby, provide multi-targeting ability to CAR T cells. In addition, secreted BiTE may recruit and interact with bystander T cells and employ these cells to act against target cells. Therefore, this method can be beneficial for efficiently targeting heterogeneous solid tumor cell populations and for circumventing their immune escape (Fig. 3G). Currently, this approach is being developed for the treatment of glioblastoma [75], a heterogeneous tumor with numerous overlapping cell clusters that exhibit distinct gene and surface marker expression patterns. Wild-type EGFR is an antigen that is frequently overexpressed in glioblastoma, but it is also expressed in normal tissues. Hence, its targeting may lead to off-target toxicity. EGFR variant III (EGFRvIII) is a mutated antigen expressed in a subset of glioblastomas, but not in non-malignant tissues. Several studies in which EGFRvIII was targeted using anti-EGFRvIII–CD3 BiTE [86] or EGFRvIII directed CAR T cells [87–90] have shown promising results. However, immune escape has also been reported after CAR T cell infusion, due to the heterogeneity of glioblastoma. To address this challenge, CART.BiTEs were developed by generating a bi-cistronic gene construct that permits co-expression of anti-EGFRvIII CARs and BiTE targeting wild-type EGFR in T cells. By applying this method, not only EGFRvIII positive tumor cells were targeted, but the CAR T cells could also release anti-EGFR BiTEs locally, recruiting and redirecting bystander T cells against tumor outgrowths that expressed wild-type EGFR [75]. Consequently, the aforementioned study showed that these CAR T cells can mediate potent and specific anti-tumor activity against heterogeneous tumor cell populations and mitigate the effect of antigen loss. Undoubtedly, local BiTE secretion enables the targeting of various and/or less specifically expressed antigens [75].

5.9 Inhibitory CARs

Another alternative to reduce undesired off-target effects is the application of inhibitory CARs (iCARs). Inhibitory CAR T cells are activated by tumor antigens, whereas normal cells are protected from a CAR T cell-mediated attack. Inhibitory CAR T cells have a receptor for normal cell-specific antigens in addition to their receptors against tumor antigens. The recognition of normal cell-specific antigens by CAR T cells triggers negative signaling endowed by inhibitory domains that have replaced the costimulatory domain (Fig. 3H) [91]. Inhibitory signaling domains are usually derived from inhibitory molecules, such as programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In a preclinical study, it was shown that iCARs feasibly harness natural T cell inhibition exerted by PD-1 and CTLA-4 while protecting normal tissues from off-target effects [92, 93]. Hence, it seems that specific immune checkpoint inhibition of normal tissue antigens can prevent off-target effects. However, a main drawback is that this inhibitory signal can entirely deactivate T cell functionality. In this regard, CD28 is now one of the most commonly used molecules that improves T cell activity via IL-2 secretion [92, 93].

5.10 Affinity-tuned CAR T cells

There is a partial overlap in antigens expressed on malignant and non-malignant cells. However, different expression levels between cancer and normal cells can be applied to true-tumor targeting. Affinity-tuned CARs can distinguish tumors with high-density antigen expression from non-malignant cells with low-density antigen expression. The higher density of tumor antigens on tumor cells makes them targetable for CARs with lower-affinity scFvs (Fig. 3F) [94]. Therefore, CAR affinity tuning may be a suitable approach for reducing off-tumor toxicity. CAR affinity optimization can be achieved by the introduction of mutations or by recombining heavy and light chains [95]. In a therapeutic study on multiple myeloma (MM), an affinity-tuned CAR T cell approach was used to target CD38 [95]. CD38 is expressed at high levels on all MM cells, and is expressed at low levels on several hematopoietic cells, including natural killer (NK) cells, monocytes and a subset of T cells. In this study, a light-chain exchange technology was used to generate a large panel of scFvs with a wide range of affinities for a CD38 epitope. CAR T cells bearing scFvs derived from low affinity antibodies showed significant anti-tumor cytotoxicities with minimal off-tumor effects.

The development of FDA-approved CD19 CAR T cells [96] and the adequate clinical responses observed in hematological malignancies suggest that CAR T cells may also be used as therapeutic candidates for solid tumors [97–99]. However, several complications restrict CAR T cell efficiency in solid tumor therapy (Fig. 4). The successful use of CAR T cell therapy in hematological malignancies is attributed to various factors, including a high expression of surface markers on leukemic cells and an accessibility of immune cells to lymphoid cells. The scarcity of homogeneous tumor-specific antigens is, however, a major obstacle for CAR T cell therapy in solid tumors [100]. Also, several recognized tumor antigens are located inside the cells instead of outside on their surface. Additionally, after intravenous administration, the first hurdle facing CAR T cells is their efficient traveling through the blood stream and homing within the tumor site. Additional barriers include CAR T cell infiltration in the rigid tumor extracellular matrix (ECM), its dominance in the immunosuppressive TME and the persistent activity of T cells. This complexity highlights the need for strategies that improve T cell trafficking, counteract the immunosuppressive TME, enhance the persistence and activity of CAR T cells and, lastly, identify true tumor-specific targets.

6.1 The absence of definitive markers in solid tumors

As mentioned above, the identification of specific tumor antigens is a substantial step in the design of CAR T cells. In this respect, solid tumors face more complications because of their intratumoral and intertumoral heterogeneity. Ample research has focused on targeting tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) [101], disialoganglioside GD2 [102], mesothelin [103], interleukin 13 receptor (IL-13R) [104], human epidermal growth factor receptor 2 (HER2) [105] and L1 cell adhesion molecule (L1CAM) [106]. TAAs are overexpressed on tumor cells and exhibit a low expression in normal cells [107]. Thus, their expression may increase the risk of off-tumor toxicity. All of the multi-target approaches discussed above, such as bi-specific CAR T cells, synNotch CAR T cells and switchable forms of CAR T cells, i.e., Zip CARs, are applied to overcome the limitations of specificity and off-tumor toxicity (Fig. 4A). Neoantigens or tumor-specific antigens (TSAs) represent another class of antigens that are considered for targeting in immunotherapy. Neoantigens are derived from novel patient-specific somatic mutations that occur during tumor development and, thus, are expressed in malignant tissues exclusively. These markers are considered as ideal targets for immunotherapy, since they allow the precise targeting of malignant cells without impairing normal cells. Personalized vaccines [108, 109] and TIL-based adoptive T cell therapies [110, 111] are two main approaches in immunotherapy that target neoantigens and have shown successful results in solid tumors. Several CAR T cell studies regarding neoantigens have been reported [112, 113]. As mentioned above, EGFRvIII is a truncated form of EGFR lacking a significant portion of the extracellular ligand-binding domain, thereby increasing the activity of the ligand-independent component [114]. EGFRvIII is encountered in a high percentage of glioblastomas. This surface neoantigen is specifically expressed on malignant cells at a high level and plays an important role in its tumorigenesis and invasiveness. In a recent clinical trial, anti-EGFRvIII CAR T cells were used against glioblastoma and showed effective results in tumor regression. No evidence for off-tumor toxicity, CRS and/or cross-reactivity to non-mutant EGFR was observed [90, 115, 116]. The majority of the recognized neoantigens are, however, located intracellularly and are immunogenic only when present in a peptide/HLA complex. Hence, for the most part they are not considered as appropriate targets for CAR T cell therapy, since conventional CARs are designed to bind to surface markers in a HLA-independent manner [117]. A recent animal study showed that this drawback can be overcome by implementing scFvs with a targeting ability of epitope/HLA complexes in CAR constructs [118], but the exact efficiency of this approach remains to be determined. In addition, a large fraction of human tumor-associated mutations are not shared between patients at meaningful frequencies and, therefore, they shoul be considered patient-specific. Detecting and targeting patient-specific mutations is, therefore, challenging.

Alternative antigens that prominently affect solid tumor treatment may be derived from the tumor microenvironment rather than from the tumor cells themselves. In contrast to tumor-specific antigens (TSAs), these antigens are shared between solid tumors and are, therefore, not patient-specific [119]. For example, cancer-associated stromal cells (CASCs) that support tumorigenesis and its related angiogenesis are potential candidates for this purpose [120]. Cancer-associated fibroblasts (CAF) represent a subset of CASCs with a significant impact on the immunosuppressive TME. These cells play an important role in tumor progression, metastasis and angiogenesis via the secretion of various cytokines, such as stromal cell-derived factor 1 (SDF1), also known as C-X-C motif chemokine 12 (CXCL12) and vascular endothelial growth factor (VEGF) [120]. Another example is fibroblast activation protein (FAP), which is mainly expressed on CAFs in various solid tumors. It has been reported that CAR T cells directed against FAPs can reduce FAP + fibroblastic cells and decrease extracellular matrix proteins and glycosaminoglycans, thereby effectively facilitating CAR T cell infiltration into the tumor matrix. Many studies have shown the effect of tumor vasculature on the growth of multiple types of cancer [121, 122]. In a xenograft tumor study, CAR T cells were designed to target VEGFR-1 and the results obtained indicated decreased tumor growth and suppressed pulmonary metastasis [106]. In addition, E3B, a splice variant of fibronectin with expression in the neovascular network of tumors, may serve as a target of CAR T cells with an inhibitory effect on tumor growth [120]. Thus, prevalent microenvironmental factors among various tumors turns them into potential candidates for therapeutic targeting.

6.2 CAR T cell delivery to the tumor site

One of the main obstacles in tackling solid tumors is the trafficking of CAR T cells to the tumor site, their infiltration into the tumor parenchyma and their homing. Immune cell trafficking to solid tumors occurs under the guidance of chemotactic signals that are produced by tumor cells or stromal cells in the TME. In response to tumor secreted chemokines, normal T cells do not express adequate chemokine receptors for traveling towards tumor sites [123]. Hence, the insertion of chemokine receptors in CAR T cells helps in sensitizing CAR T cells to chemokine gradients formed by tumoral and/or associated stromal cells (Fig. 4B). The various examples of chemokine receptors in CAR T cell designs include C-C motif chemokine receptor (CCR)2 [124], CCR2b [124, 125], CCR4 [126], C-X-C chemokine receptor (CXCR)1 [127], CXCR2 [128, 129] and CXCR3 [129]. There are, however, some drawbacks regarding the efficiency of these engineered CAR T cells. In inflammation or injury conditions, for example, other organs also produce chemokine gradients that may interfere with CAR T cell trafficking to the tumor site [130]. Also, following CAR T cell trafficking in response to specific chemokine gradients, immune escape may occur due to chemokine loss or down-regulation [130]. In addition, the landscapes of factors secreted by tumors are extremely heterogeneous and vary in both intratumoral sites and between individuals [131].

Another novel strategy for counteracting heterogeneous and inadequate tumor chemokines is inducing the expression of chemokine ligands in tumor cells. Therefore, various intratumoral delivery methods, such as adenoviruses expressing C-C motif chemokine ligand (CCL)17 [132] or plasmids encoding chemokine ligand CCL5, have been tested [133]. Although no safety obstacles were identified in the intratumoral delivery methods, this approach is not feasible in at least some patients due to inadequate tumor position, technical difficulties and/or complications in identifying metastatic locations. One approach for circumventing the obstacle of T cell infiltration is regional/local CAR T cell administration. Administering CAR T cells directly into the tumor site may also diminish the risk of off-tumor toxicity [134]. Regional delivery appears particularly appealing for CAR T cell therapy of central nervous system (CNS) tumors, due to the safe delivery of CAR T cells into the cerebrospinal fluid. Preclinical studies have also shown that lower doses are needed in this approach, compared to the systemic administration of CAR T cells [135, 136]. Although direct infusion of CAR T cells has been reported in various anatomical sites, such as brain [137], breast [138] and liver [139], this method is not feasible in some tumors and, in addition, is accompanied by technical limitations. The local CAR T cell infusion approach may be limited to the initial tumor site, without a direct effect on metastatic lesions. Locally infused CAR T cells may, however, initiate and promote endogenous immune responses.

Tissue infiltration of T cells is a multistep process, including the adhesion of molecules on T cells and vasculature cells, and the rolling and the extravasation of circulating T lymphocytes into the tissue [132]. Intercellular adhesion molecule (ICAM) [131], L-selectin [140], vascular cell adhesion protein (VCAM) [141] and other ligands and receptors involved in the aforementioned processes are promising targets for CAR T cell therapy. Physical barriers, including the fibrotic ECM, can impede T cell penetration. Therefore, CAR T cells that induce ECM modifying enzyme expression may facilitate CAR T cell infiltration (Fig. 4C). In an in vivo study it was, for instance, shown that GD2-directed CAR T cells expressing heparinase could degrade heparin sulfate proteoglycans in the ECM, thus enhancing CAR T cell infiltration [142]. In many cases, the physical characteristics of CARs may play an important role. It has been found, for instance, that using nanobodies instead of scFvs can facilitate CAR T cell infiltration into the TME. Nanobody-based CAR T cells only contain the variable fragments of the heavy chains in their antigen-binding site. The small size of the nanobodies helps in penetrating the blood-brain barrier and solid tumor matrixes and in identifying markers that are not accessible for large antibodies [16].

6.3 Immunosuppressive microenvironments

After crossing the barriers towards the tumor site, CAR T cells encounter the immunosuppressive TME. Abundant immune suppressor cells including regulatory T cells (Tregs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) [143] are located in the TME. These cells moderate anti-tumor activity by secreting various immunosuppressive cytokines, such as TGF-B, IL-4, and PD-L1. These factors inhibit immune cell function and activation, thus limiting the therapeutic potential of cancer immunotherapy [144]. A selective depletion of immunosuppressive cells in combination with CAR T cell therapy can increase CAR T cell efficacy [145]. It has been found, for instance, that combination of Treg depletion with systemic CAR T cell therapy enhances anti-tumor activity in preclinical solid tumor models. The efficacy was attributed to decreased immunosuppressive cytokines, including TGF-B, PD-1 and IL-10 that are secreted by Tregs [146]. Depleting MDSCs can also boost CAR T cell responses. In a xenograft sarcoma model, it was shown that the employment of GD2 CART cells together with MDSC depletion led to significant anti-tumor efficacy, whereas CAR T cells alone induced a lower anti-tumor activity [145].

Alternatively, various cytokines secreted by immune cells are considered as suitable targets for CAR T cells. In a recent prostate cancer study, it was shown that engineered prostate-specific membrane antigen (PSMA) CAR T cells that co-express a dominant-negative TGF-RII exhibited an increased proliferation rate and a long-term in vivo persistence, and could eradicate the tumor [145]. These promising results in preclinical models have led to a clinical trial in patients diagnosed with relapsed and refractory metastatic prostate cancer (NCT03089203).

PD-1 is one of the best characterized immune inhibitory molecules present in the TME. This molecule has been studied as a potential target that can enhance CAR T cell efficacy, Several strategies have been used to manipulate PD-1 expression on CAR T cells: (i) Numerous studies have demonstrated that co-administration of PD-1/PD-L1 blockers with CAR T cell therapy enhances anti-tumor activity and prevents T cell exhaustion in preclinical models [147] and patients receiving mesothelin CAR T cell therapy [141]. Notably, systemic blockade of PD-1/PD-L1 may be accompanied by undesirable effects, generating systemic checkpoint blockade in diseases such as pneumonitis, colitis and hepatitis. Nevertheless, checkpoint inhibition may cause T cell toxicity because of limited T cell safety. (ii) Genetic knockout of PD-1 prevents the adverse side effects of systemic removal of PD-1. The CRISPR/Cas9 method has been used in several in vitro and in vivo xenograft studies, exhibiting enhanced anti-tumor efficiencies [148]. CTLA-4 [149] and lymphocyte activation gene 3 (LAG3) [150] are other checkpoint molecules that have been efficiently deleted by CRISPR/Cas9, yielding T cells that are less prone to checkpoint inhibition and exhaustion, hence resulting in improved anti-tumor efficacies and persistence in preclinical models. (iii). Armored CAR T cells have been genetically modified to secrete PD-1 suppressors at the tumor site. As a result of the PD-1 blockade, increased anti-tumor efficacy was observed (Fig. 4D) [151]. In another approach, the TME can be used against the tumor itself. In other words, modified CAR T cells can switch immune inhibitory signals in the TME to stimulatory signals. For this purpose, the extracellular segments of PD-1 are fused to the costimulatory domains in the CAR design. Subsequent CAR T cell attachment to PDL-1 leads to CAR T cell activation (Fig. 4E) [152]. This approach can be similarly used to convert other inhibitory signals in the TME into proinflammatory signals. Functional CAR T cells have, for instance, been engineered by linking the extracellular portion of inhibitory molecules, such as IL-4 receptors, to the endodomains of proinflammatory cytokines, such as IL-7, IL-2 and IL-15. As a result, immune inhibitory cytokines behave like immune stimulatory agents [153].

In order to subdue the immunosuppressive characteristics of the TME for immune cell activity, several studies have employed armored CAR T cells, also known as TRUCKs, with the capacity to induce several stimulatory cytokines, such as IL-12 [152], IL-7 [154], IL-15 [100] and IL-21 (Fig. 4F) [155]. It was found that these cytokines can enhance CAR T cell immunity by reducing the effects of immunosuppressive agents in the TME, as well as by increasing the expansion and persistence of CAR T cells. In a preclinical model of ovarian cancer, for instance, IL-12-armored CAR T cells were used directed against MUC-16^ecto antigen [156, 157], resulting in an increase in anti-tumor activity, proliferative capacity, survival and toxicity. These CAR T cells exhibited higher persistence and lower exhaustion. The results obtained in the latter study have led to a clinical trial (NCT02498912) in which IL-12-armored CAR T cells targeted MUC-16^ecto are used for the treatment of patients suffering from ovarian cancer [158]. Recently, it has been reported that modified CAR T cells with inducible IL-18 can enhance proinflammatory cytokines and direct immune cell function towards cytotoxicity versus immune-regulatory, thereby decreasing the infiltration and homing of immunosuppressive cells such as Tregs and CD206 + macrophages, and boosting immune attacks [159, 160].

The production of CAR T cells includes several precise steps that start with obtaining peripheral blood mononuclear cells (PBMCs) from patients, commonly via leukapheresis. Subsequently, enriched T cells from the leukapheresis product are activated, transduced by a CAR transgene, and expanded. After successfully passing quality control tests, CAR T cell products are infused into patients. Although the general outline of CAR T cell-based clinical trials are similar, each of the steps involved is carried out by a variety of approaches [161]. Several T cell subsets can be isolated for CAR T cell manufacturing. In the majority of clinical trials, the CD3 + population of T cells is used [44, 162]. In some clinical trials, however, other T cell subsets such as naïve cells [163], memory stem cells [163] and central memory cells [164] have been applied for CAR T cell manufacturing. Different tools, such as CliniMACS, Plus and Prodigy, have enabled the selection of certain subsets of T cells based on different surface markers, such as CD4, CD8, CD25 and CD62L [165]. T cell activation is the next step in CAR T cell manufacturing, which is required for sequential expansion and transduction [165]. Three main methods for T cell activation are used in CAR T cell clinical trials. Monoclonal antibodies, such as anti-CD3, in the presence of various interleukins, such as IL-2 [166], IL-15 [167] and IL-7 [167], can generally activate patient-derived T cells, independent of specific antigens. In some clinical trials, feeder cells, including autologous [45] or allogeneic [168] PBMCs, are used to support T cell activation and expansion. In another approach, artificial antigen-presenting cells (aAPCs), such as K562 cells, are engineered to express co-stimulatory molecules, such as CD32, CD64, CD86 and CD13, on their cell surface to activate T cells [169]. K562 cells lack HLA expression and are, therefore, unlikely to activate alloreactive T cells [170]. A more efficient and potent method, which is frequently used in clinical CAR T cell manufacturing, is independent of antigen presentation and involves stimulation of T cells with paramagnetic 4.5-µm diameter beads, which are covalently coated with anti-CD3/anti-CD28 mAbs, along with IL-2 supplementation [171]. The next challenge in CAR T cell manufacturing is successive gene delivery that leads to stable CAR expression. To this end, various viral and non-viral systems for CAR transfer have been developed, including retro- and lentiviral vectors, as well as transposon/transposase and mRNA transfer-mediated gene expression systems [161]. The high transduction efficiency and CAR expression stability allegedly derived from retro- or lentiviral transfer systems, seem to be the main reasons why these gene delivery approaches are commonly applied in clinical CAR T cell manufacturing processes [172]. However, several drawbacks such as cumbersome production scale-up, intensive and expensive good manufacturing practices (GMP) required for production and the risk of oncogenic insertions, restrict the use of viral vectors. Hence, alternatives such as the transposon/transposase system have been evaluated in various studies. In comparison to the viral transfer systems, these systems profit from less complicated manufacturing procedures and are economically advantageous [173]. The main drawback may, however, be the oncogenic nature of the transposon/transposase system. In contrast to stable CAR expression by the viral and transposon/transposase systems, in the CAR mRNA transfection system the relatively short lifespan of mRNA leads to transient cytoplasmic CAR expression. In a mesothelin-targeted CAR T cell therapy study, long-term expression of CAR was provided by sequential infusions of mRNA-transduced CAR T cells [174]. Subsequent injections were carried out in multiple series, followed by toxicity assessments in each step. This approach may be beneficial for toxicity management. To obtain a therapeutic dosage of transduced CAR T cells, they must be expanded. Subsequently, quality control tests must be conducted for checking various criteria, such as identity, safety, purity and potency [161, 165].

In addition to the enormous variation mentioned above, one of the most important variations is related to the availability of targetable tumor markers. Due to the heterogeneous character of solid tumors, there are more variations in solid tumor markers than in hematological tumor markers. CD19 is the most common marker in hematological malignancies, which can be tackled by CAR T cell therapy. Additional tumor-related targets are being studied in phases I and II leukemic clinical trials, including CD20, CD137, CD28, CD33 and B-cell maturation antigen (BCMA). Similarly, various markers such as CD171, PSMA, IL13Rα2, CEACAM5, HER2, GD2, CAIX, mesothelin, EGFRvIII, Claudin 18.2 and NKG2D are being studied in solid tumors (Tables 1 and 2).

Table 1

Major clinical trials of CAR T cell in solid tumors.
Disease	Target /Construct	Gene delivery system	Activation	CAR generation	Response	Phase/ Clinical Trials identifier	Toxicity	Reference/ Year
Neuroblastoma	Anti-CD171	Plasmid, electroporation	OKT3, IL-2	I	Nr	I/ Nr	Nr	[178]/2007
Neuroblastoma	Anti-GD2	Retroviral transfection	OKT3, IL-2	I	Nr	I/ NCT00085930	Nr	[179]/2011
Glioblastoma	Anti-IL13Rα2/ an interleukin-13 (E13Y-mutated) ligand-based CAR containing a 4-1BB costimulatory domain (IL13BBζ), and truncated CD19 (CD19t)	Intracranial injection	Nr	II	sustained clinical response for 7.5 months after the initiation of CAR T-cell therapy, and none of these initial tumors (tumors 1 through 7 and spinal tumors) recurred	I/ NCT02208362	No toxic effects of grade 3 or higher, Grade 1 or 2 events were observed	[180, 181]/ 2016
Prostate Cancer	Anti-PSMA	Retroviral transfection	IL-2	I	Two-of-five (40%) patients achieved clinical PR, with PSA declines of 50% and 70% and PSA delays of 78 and 150 days, plus a minor response in a third patient.	I/ Nr	No anti-PSMA toxicities were observed	[182]/ 2016
Colon cancer	Anti- ERBB2 (HER-2/neu)/ CAR vector containing CD28, 4-1BB, and CD3ζ signaling moieties was assembled in a γ-retroviral vector (MSGV1-4D5-CD8-28BBZ)	Retroviral transfection	Anti-CD3 mAb and interleukin-2 (IL-2)	III	Increases in interferon-γ (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10, consistent with a cytokine storm	I/II / NCI-09-C-0041	Patient was died	[183]/ 2010
CEACAM5 + tumors	Anti-CEACAM5 / CEACAM5-specific scFv (MFE23) fused to CD3ζ	Antibody-directed pro- drug therapy strategies	IL2	I	No objective clinical responses were observed	I/ NCT01212887	Respiratory toxicity was observed	[184]/ 2017
Renal cell carcinoma	Anti- carboxy- anhydrase-IX (CAIX)	Retroviral transfection	human recombinant interleukin-2	I	No clinical responses were observed	I/ DDHK97-29/P00.0040C	no liver toxicities were observed	[185]/ 2013
Colorectal cancer	Anti-NKG2D receptor/ NKG2D ligand and TCR signaling	Retroviral transfection	Nr	Nr	Nr	I/ NCT03310008	Grade3/4 related AE and graft-versus-host disease (GvHD) occurrence were observed	[186]/ 2019
Pancreatic ductal adenocarcinoma	Anti-Mesothelin	mRNA electroporation	Nr	Nr	The total metabolic active volume (MAV) remained stable in 3 patients and decreased by 69.2% in 1 patient. In this patient, all liver lesions had a complete reduction in FDG uptake at 1 month, there was no effect on the primary PDAC.	I/ Nr	None of the patients developed CRS or neurologic symptoms and there were no dose-limiting toxicities.	[187]/ 2018
Recurrent glioblastoma	Anti- the epidermal growth factor receptor variant III (EGFRvIII)/ a lentiviral construct with 4-1BB costimulatory domain ( humanized anti-EGFRvIII single-chain variable fragment fused to the hinge and transmembrane domain of CD8 and the human 4-1BB and CD3ζ intracellular signaling domains)	Intravenous infusion	Nr	II	Nr	I/ NCT02209376	No subjects experienced evidence of EGFR-directed toxicity or systemic CRS	[188]/ 2017
Recurrent ovarian cancer	Anti- MUC-16 ecto/ 4H11 CAR Binding Domain and MUC16 ecto ( 4H11-28z/fIL-12/EFGRt)	intravenous and intraperitoneal injection	IL-12	II	Nr	I/ NCT02498912	Severe CRS was observed	[189]/ 2015
Glioblastoma	Anti-EGFRvIII/ EGFRvIII CAR containing CD28 and 4–1BB costimulatory domains, and CD3ζ signaling domain	Intravenous injection	IL-12	III	One patient with survival of 59 months	I / NCT01454596	Two patients experienced severe hypoxia.	[190]/ 2019
Glioblastoma	Anti-HER2/ HER2-CAR with an FRP5-based exodomain and a CD28.ζendodomain	Intravenous injection	IL-2	II	One PR was observed. Eight Patients had a clinical benefit, with a median overall survival of 11.1 Months after T-cell infusion and 24.5 months after diagnosis, and 3 patients were alive with no disease progression at the last follow-up.	I/ NCT01109095	This phase1dose-escalation study established the safety of autologous HER2-CAR T in17 patients with Progressive glioblastoma with no serious AE.	[191]/ 2017
Breast Cancer	Anti-c-Met/ c-Met, BBZ CAR construct	Intratumoral injection	Nr	II	Intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors.	0/ NCT01837602	None of the patients had study drug-related AE greater than grade 1.	[192]/ 2017
Metastatic lung and breast cancers	Anti-Mesothelin/ CD28-costimulated MSLN CAR T cells with the I-caspase-9 safety gene	Intra pleural injection	Nr	II	Best responses included 2 patients with the complete metabolic response on PET (62 and 39 weeks ongoing); 5 PR	I/ NCT02414269	No CAR T-cell–related toxicities higher than grade 1 were observed. 1 patient developing grade 3 pneumonitis	[193]/ 2019
Metastatic Colorectal Cancers	Anti- CEA+/ BW431/26 scFvIgG4 hinge-CD28 transmembrane domain-CD28-CD3z	Plasmid transfection	IL-2	II	Most patients had an apparent decrease in the CEA levels as response to CAR-T therapy, especially in long-term observation.	I/ NCT02349724	Severe AE related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced PD in previous treatments had stable disease after CAR-T therapy	[194]/ 2017

Table 2

Major clinical trials of CAR T cells in hematological malignancies.
Disease	Target /Construct	Gene delivery system	Activation	CAR generation	Response	Phase/ Clinical Trials. gov identifier	Toxicity	Reference/ Years
MCL	CD20/Plasmid Vector including a CD20-specific chimeric T-cell receptor and neomycin resistance gene	Electroporation	OKT3, IL-2, irr.all. PBMCs, LCL	1st	28.57% maintained a previous complete response 14.29% achieved a partial response 57.14% stable disease	I/ NCT00012207	25% a flulike syndrome, 25% fever, skin reactions at injection site, chills, myalgias, dyspnea, dysgeusia, malaise or fatigue, diaphoresis, and injection site reactions was observed	[195]/ 2008
AML	CD33/Lentiviral vector harboring anti-CD33 scFv, human CD8α hinge and transmembrane domains, and human 4-1BB and CD3ζ signaling domains	Lentivirus vector	antihuman CD3 monoclonal antibody, recombinant human IL-2, recombinant human interferon (IFN)-γ	1 st	Nr	I/II/ NCT01864902	The CART-33 infusion alone induced rigorous chills and fevers; drastic fluctuations of his preexisting pancytopenia; elevated serum cytokine levels, including IL-6, IL-8, tumor necrosis factor-α, and interferon-γ; slight transient hyperbilirubinemia within 2 weeks was observed. a subsequent intermittent moderate fever; and reversed fluctuation of the pancytopenia was observed.	[196]/ 2015
DLBCL	CD19/ Lentivirus vector harboring anti-CD19 scFv, human 4-1BB and CD3ζ signaling domains	Lentiviral vector	Nr	2 st	82% OR, 73% CR, 9% PR and 18% PD	I/ NCT02631044	One DLBCL patient in CR had a parenchymal brain lesion in the right temporal lobe that also completely resolved. Of note, this patient had no CRS or neurotoxicity associated with JCAR017 treatment	[197]/ 2016
DLBCL	CD19/Lentivirus vector harboring anti-CD19 scFv, human 4-1BB and CD3ζ signaling domains	Lentiviral vector	Nr	2 st	Among patients evaluable for efficacy (n = 255), ORR was 73% (95% CI, 67‒78) the CR rate was 53% (95% CI, 47‒59). Responses were similar across all patients subgroups. Median DOR was 13.3 months (95% CI, 8.2‒not reached [NR]) with 10.8 mo of median follow-up. Median DOR for patients in CR was NR (13.3‒NR). Median PFS was 6.8 months (95% CI, 3.3‒11.8).	I/ NCT02631044	Safety analysis showed 79% of patients had grade ≥ 3 AEs, primarily cytopenias (neutropenia, 60%; anemia, 37%; thrombocytopenia, 27%). CRS or NE occurred in 47% of patients was observed. Any grade CRS occurred in 42% of patients at a median onset of 5 days; only 2% had grade ≥ 3 CRS was observed. NEs observed in 30% of patients (grade ≥ 3, 10%) at a median onset of 9 days	[198]/ 2019
DLBCL	C19/ retroviral vector containing the CAR gene construct	Retrovirus	anti-CD3 and IL-2	2 st	The overall response rate was 71% (n = 5/7) and complete response (CR) rate was 57% (n = 4/7).	I/II/ NCT02348216	Grade R3 14% CRS and 57% neurotoxicity was observed. All other KTE-C19-related grade R3 events were resolved within 1 month.	[199]/ 2017
DLBCL	C19/ retroviral vector containing the CAR gene construct	Retrovirus	anti-CD3 and IL-2	2 st	The objective response rate was 82%, and the complete response rate was 54%. With a median follow-up of 15.4 months, 42% of the patients continued to have a response, with 40% continuing to have a complete response. The overall rate of survival at 18 months was 52%.	II/ NCT02348216	The most common adverse events of grade 3 or higher during treatment were neutropenia (in 78% of the patients), anemia (in 43%), and thrombocytopenia (in 38%). Grade 3 or higher cytokine release syndrome and neurologic events observed in 13% and 28% of the patients, respectively. Three of the patients died during treatment.	[200]/ 2017
ALL	C19/CD3ζ and co-stimulatory signals (4-1BB)	Lentivirus	intracellular immunoreceptor tyrosine-based activation motifs	2 st	83% achieved CR/CRi within 3 months (all had minimal residual dis-ease negative marrow). Relapse-free probability at 6 months was 75% (95% CI, 57%-87%), probability of 6-month survival 89% (95% CI, 77%-94%).	II/ NCT02435849	CRS observed in 78% of patients (21% grade 3; 27% grade 4) 49% of whom received tocilizumab. No deaths due to refractory CRS observed. Other grades 3/4 non-hematologic AEs (> 15%) were hypotension (22%), and hypoxia (18%). 15% of patients experienced grade 3 neurological AEs, with no grade 4 events and no cerebral edema reported. 60% had grade 3/4 neutropenia with high fever (> 38.3C). 2 patients died within 30 days of infusion; 9 patients died > 30 days after infusion.	[201]/ 2017
DLBCL	C19/CD3ζ and co-stimulatory signals (4-1BB)	Lentivirus	intracellular immunoreceptor tyrosine-based activation motifs	2 st	95% of CRs was observed in at 3 months.	II/ NCT02445248	86% of patients had grade 3 or 4 AEs. CRS observed in 58% of infused patients, with 15% grade 3 and 8% grade 4. Other grades 3 or 4 AEs of special interest included neurologic AEs (12%, managed with supportive care), cytopenias lasting > 28 days (27%), infections (20%), and febrile neutropenia (13%). Three patients died within 30 days of infusion, all due to disease progression. No deaths were attributed to CTL019. No CRS or neurologic event associated deaths observed.	[202]/ 2017
Lymphoma	CD19/gamma-retroviral vector and contained an anti-CD19 single-chain variable fragment derived from a murine monoclonal antibody, hinge and transmembrane regions from human CD28, the CD28 costimulatory domain, and the CD3ζ T-cell activation domain	Retroviral vector	the anti-CD3 monoclonal antibody OKT3and IL-2	2 st	The overall remission rate was 73% with 55% complete remissions and 18% partial remissions.	I/II/ NCT00924326	Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that were completely resolved.	[203]/ 2017
MCL	CD19/retroviral vector harboring an anti-CD19 scFv, CD3-zeta T-cell activation domain and CD28 signaling domain	Retroviral vector	Nr	1 st	The primary efficacy analysis showed that 93% (95% confidence interval [CI], 84 to 98) of the 60 patients in the primary efficacy analysis had an objective response; 67% (95% CI, 53 to 78) had a complete response. In an intention-to-treat analysis involving all 74 patients, 85% had an objective response; 59% had a complete response.	II/ NCT02601313	Common adverse events of grade 3 or higher were cytopenias (in 94% of the patients) and infections (in 32%). Grade 3 or higher cytokine release syndrome and neurologic events observed in 15% and 31% of patients, respectively; none were fatal. Two grade 5 infectious adverse events observed.	[204]/ 2020
ALL	CD19/retroviral vector harboring an anti-CD19 scFv, CD3-zeta T-cell activation domain and CD28 signaling domain	Retroviral vector	Nr	1 st	Among the 32 patients evaluable for response, the overall rate of undetectable MRD was 78% (95% CI, 60–91%). CR or CRi was achieved by 23 patients (72%), and 1 patient (3%) had blast-free bone marrow.	I/ NCT02614066	There was 2 KTE-X19-related Grade 5 events: 1 cerebral infarction at the 0.5 × 10⁶ cells/kg dose and 1 previously reported multiorgan failure secondary to CRS at the 2 × 10⁶ cells/kg dose. Grade ≥ 3 neurologic events observed in 16 patients (46%, median time to onset 7 days [range, 4 − 24 days]). Neurologic events resolved in all patients (excluding 2 patients with unresolved events at death), with a median time to resolution of 17 days (range, 6 − 53 days).	[205]/ 2019
ALL	CD19/retroviral vector harboring an anti-CD19 scFv, CD3-zeta T-cell activation domain and CD28 signaling domain	Retroviral vector	Nr	1 st	Of 41 patients with ≥ 2 months of follow-up, 68% had CR/CRi, and 73% had undetectable minimal residual disease. 84% CR/CRi, was observed and the median event-free survival was 15 months.	I/ NCT02614066	The most common Grade ≥ 3 AEs were hypotension (38%), pyrexia (38%), and thrombocytopenia (31%). Grade ≥ 3 CRS and NEs observed in 13 (29%) and 17 (38%) patients, respectively.	[206]/ 2019
ALL	CD19/retroviral vector harboring an anti-CD19 scFv, CD3-zeta T-cell activation domain and CD28 signaling domain	Retroviral vector	Nr	1 st	The CR + CRi rate was 100%, 64%, and 71% in the 2 × 10⁶, 1 × 10⁶ (68-mL), and 1 × 10⁶ (40-mL) groups, respectively, with 25%, 71%, and 100% of CR + CRi patients in ongoing response as of the data cutoff; and 75%, 73%, and 86% of all patients had undetectable minimal residual disease.	I/ NCT02625480	The most common Grade ≥ 3 adverse events were hypotension (50%) and anemia (33%). Rates of Grade ≥ 3 neurologic events were 25%, 36%, and 11% in the 2 × 10⁶, 1 × 10⁶ (68-mL), and 1 × 10⁶ (40-mL) groups, respectively, and rates of Grade ≥ 3 cytokine release syndrome were 75%, 18%, and 22%. Overall, 3 Grade 5 adverse events were unrelated to KTE-X19.	[207]/ 2019
DLBCL	CD19/retroviral vector harboring an anti-CD19 scFv, CD3-zeta T-cell activation domain and CD28 signaling domain	Retroviral vector	Nr	1 st	Best overall and complete response rates in infused patients were 82% (95% CI, 77–86%) and 64% (95% CI, 58–69%), respectively. At a median follow-up of 12.9 months from the time of CAR T-cell infusion, median progression-free survival was 8.3 months (95% CI, 6.0 to15.1 months), and median overall survival was not reached.	Nr / NCT03153462	Among the axi-cel–treated patients, grade 3 cytokine release syndrome and neurotoxicity observed in 7% and 31%, respectively. Non-relapse mortality was 4.4%.	[208]/ 2020
B-cell lymphoma relapse	CD19/retroviral third-generation CAR Vector (RV-SFG. CD19.CD28.4-1BBzeta) that comprises CD137 (4-1BB) and CD28 as costimulatory domains	Retroviral vector	Nr	2st or 3 st	6 of 8 (75%) of the patients responded	I/ NCT03676504	CAR treatment was well tolerated with grade ≥ 3 cytokine release syndrome and neurotoxicity each being observed after 1 of 10 dosings. A single patient had moderate chronic GVHD. Of note, three of four patients who received axi-cel had ongoing grade ≥ 3 cytopenia three months post-dosing, whereas prolonged cytopenia was not observed in 9 alloHCT-naïve patients who received axi-cel during the same time period.	[209]/ 2020

To date, the FDA has approved two CAR T cell products for non-Hodgkin lymphoma therapy, i.e., axicbtagene ciloleucel (Yescarta) and tisagenlecleucel (Kymriah) [175]. Axicabtagene ciloleucel (axi-cel) is developed for the treatment of adult patients with relapsed or refractory large B cell lymphoma, after going through at least two lines of unsuccessful systemic therapy [176]. Axi-cel is generated from bulk T cells (an undefined ratio of CD4:CD8 autologous T cells) by employing a retroviral vector that includes a CD28 costimulatory domain. Subsequent T cell activation occurs in the presence of anti-CD3 antibody and IL-2. Both the potency and the safety of axi-cel products have been evaluated in a multicenter clinical trial of over 100 adults with refractory or relapsed large B cell lymphoma. The overall response rate was 83%, with 58% of the individuals achieving complete remission. At twenty-seven months of median follow-up, the progression-free survival (PFS) was 39%. Notably, a few relapses were reported six months after axi-cel infusion. The second anti-CD19 CAR T cell product, receiving FDA approval for diffuse large B cell lymphoma (DLBCL), was tisagenleclecel (tisa-cel). Tisa-cel is generated from bulk T cells by transducing CAR T cells with a CD4-1BB costimulatory domain, using lentiviral systems [177]. The overall and complete response rates were 52% and 40%, respectively. In a one year follow-up, approximately 35% of the patients indicated a progressing remission, and the overall survival of all patients undergoing tisa-cel treatment was reported to be 49%. CRS was reported in 93% of the patients after axi-cel infusion, and 13% of the cases had severe CRS (grade 3–4), which occurred at a median of 2 days after CAR T cell infusion. Tisa-cel infusion led to a 58% CRS incidence at a median of 3 days, with 11% of them identified with a severe grade of CRS [175, 177]. It is important to note that in these two studies different grading scales were applied to CRS severity. Hence, a precise comparison is impossible. It has been found, however, that both CRS and neurologic toxicity syndromes occur commonly after axi-cel infusion. It seems that the incidence and severity of toxicities in patients under axi-cel therapy may be attributed to the higher activity level of CD28 costimulatory domains in comparison to CD4-1BB. On the hand, the observed toxicities following CAR T cell infusions were reversible and manageable. The personalized nature of CAR T cell therapy and its undefined application protocol restricts widespread progression of CAR T cell therapies. Since it seems that several factors play a role, more thorough studies must be conducted to tackle the as yet unrsolved challenges.

CAR T cells have been introduced as a promising therapeutic modality for refractory hematologic malignancies. Additional clinical results suggest an efficient application of CAR T cells to the treatment of solid tumors. There are, however, also several obstacles, such as antigen escape, T cell dysfunction and toxicity that may occur after CAR T cell infusion. Next-generation therapeutics are being developed to overcome these barriers and to produce more efficient CAR T cells. CAR designs that target more than one antigen, sequentially or simultaneously, limit the venture of immune escape or off-tumor toxicity. Other investigations are adopting strategies to control the lifespan or activity of CAR T cells because of the deleterious effects that their long-term persistence and excessive activity may have. For solid tumors there are more hindrances in CAR T cell efficacy due to the impenetrable characteristics of the TME and its role in immune suppression. The fourth generation of CAR T cells has successfully overcome this obstacle by behaving like a living cargo and delivering immune-modulatory molecules to the TME, thereby altering the inhibitory milieu of the TME. The above mentioned limitations of CAR T cell therapy highlight the need for more in-depth investigations to broaden the applicability of CAR T cell therapy. For this purpose, several engineering strategies have recently been implemented. The increasing complexity of CAR T cell manufacturing methods, however, implicates that they are becoming more and more time-consuming and expensive. The personalized character of CAR T cell therapy additionally hampers precise comparisons between studies. Also, gene-editing, which is used for the production of CAR T cells, has increased the risk of tumor induction. Thus, supplementary studies must be conducted to enhance CAR T cell efficacy with minimal side effects and to attain a well-defined yet simplified protocol that enables production scale-up with reduced costs and risks.

CAR T cell therapy against B cell malignancies has exceeded expectations, leading to FDA approved CAR T cell products and several CAR T cell-based clinical trials. Due to this effective aftermath, CAR T cell therapy is considered as a promising therapeutic approach for cancer treatment. In order to optimize the efficacy of CAR T cell therapy, novel strategies have focused on rearranging and improving the structure of CAR in order to enhance its safety and specificity and decrease its on-target off-tumor toxicity. CAR T cell therapy still faces several hurdles in its application to solid tumors, including lack of tumor-restricted antigens, T cell trafficking to the tumor, immune-suppressive microenvironments and, lastly, in vivo persistence and expansion. To surmount the barriers in CAR T cell therapy for solid tumors, novel developments are aimed at structures that are: synchronically targeting several antigens, equipped by sensors that facilitate effective CAR T cell trafficking, enhanced by sensors that help distinguishing cancer cells from normal cells, and genetically modified to increase potency and resistance to immune-suppressive factors in the tumor niche. Next-generation therapeutics are poised to improve B cell therapeutics, as well as to establish these therapeutics for other malignancies, especially solid tumors.

Funding

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Conflicts of interest/Competing interests

The authors declare that they have no competing interests.

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Authors’contributions

MA and MM were the major contributors to the writing and revision of the manuscript. SSS and NF collected the related references and participated in discussions. MA and MM revised this article critically for important intellectual content. MS gave approval for the final version of the manuscript. All authors have read and approved the final manuscript.

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CAR T cell therapy as a promising approach in cancer immunotherapy: challenges and opportunities

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Abstract

Figures

1 Introduction

2 Car T Cell Therapy

3 Car T Cell Evolution

4 Challenges And Opportunities

5 Car T Cell Activity Management

6 Car T Cell Therapy Limitations For Solid Tumors

6.3 Immunosuppressive microenvironments

7 Clinical Car T Cell Manufacturing

8 Future Perspectives

9 Conclusions

Declarations

References

Status:

Journal Publication

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