Patients who are eligible for CAR T cell therapy, are deadly sick and have no further treatment options. FDA/EMA-approved CD19- and BCMA-specific CAR T cell products are mainly last line therapies, however, ironically provide high complete remission induction rates and increase overall survival in B-lineage cancers including multiple myeloma substantially (86). With excellent response rates in CD19 positive cancers, a relevant proportion of patients may experience a sustained complete response (which can be considered a cure from the primary disease) in 40% of B-ALL (4, 87) and DLBCL patients (88, 89), whereas in CLL the sustained complete response rate is 20% (90).

Despite the great success of CAR T cells, in multiple myeloma, the cancer cells’ antigen heterogeneity and immunosuppressive factors in the tumor niche have highlighted future challenges to be overcome (91). BCMA-specific CAR T cells have demonstrated significant clinical benefits in multiple myeloma patients that otherwise have no further treatment available to date (92). Yet, multiple myeloma is continuously and largely considered incurable and new treatment strategies are required to improve outcomes (93). Obviously, potent therapies justify being considered earlier in the treatment algorithm and with less than 10% of newly diagnosed multiple myeloma patients to receive CAR T cell therapy (94) because they die before being eligible according to international guidelines. In due course adjustments are required to be made carefully based on clinical outcomes. It is noteworthy that multiple myeloma patients who received the BCMA CAR T cell product idecabtagene vicleucel (bb2121) in the clinical trial KarMMa (NCT03361748) showed a median overall survival of 19.4 months (92) which represents a significant survival benefit compared to untreated patients, non-responders and patients with partial response only.

Despite significant responses in patients with various solid cancers treated with CAR T cells, such as disialoganglioside GD2 in neuroblastoma (95) and H3K27M-mutated diffuse midline gliomas (96), mesothelin (MSLN) in mesothelioma (97), claudin 18.2 (CLDN18.2) in pancreatic cancer (98), and IL13Rα2 in combination with EGFR in glioblastoma multiforme (99), CAR-based therapies for solid cancers face major limitations and challenges. Although patients may experience objective tumor regression and even complete remissions, most cancers inevitably relapse due to target antigen loss or antigen heterogeneity, which hinders primary complete responses (95, 97). Additionally, reduced CAR T cell persistence (96), the inability of T cells to infiltrate tumors, and the inhibition of CAR T-cell function by the immunosuppressive microenvironment (97) are significant barriers to durable responses. These issues can potentially be addressed by combinatorial strategies, such as integrating CAR T cells with immune checkpoint inhibition (100).

Without immediate tumor control, patients may suffer from severe cancer-induced complications or die from cancer progression. Therefore, an immediate and effective anti-cancer response is essential for a successful CAR T cell therapy (4, 101). The containment of cancer growth is facilitated by the cellular cytotoxicity, the secretion of proinflammatory cytokines and the exponential proliferation, which are triggered by the antigen-specific CAR activation with rapid kinetics inducing effector functions in alignment with T cell receptor mediated immune responses (102). Especially, the trafficking and proliferation kinetics (103) to outnumber the cancer cells and the enduring anti-cancer response by the CAR T cells until the entirety of the cancer is cleared or reduced to a molecular residual disease are key requirements to prevent cancer recurrence (104). Additionally, the CAR must have a high sensitivity in terms of recognizing and eliminating low antigen density expressing tumor cells to ensure profound cancer control (12). Otherwise, the antigen low expressing cancer cells may escape the CAR-mediated recognition whereas the antigen negative cancer cell subsets cannot be recognized by the CAR T cells and in due course initiate the relapse (91, 105). Identifying the optimal CAR sensitivity and -kinetics depend on multiple factors, including the differential antigen expression in healthy versus cancerous tissue (106), the tumor microenvironment, the level of inflammation, and the responsiveness of the specific CAR design (12, 102). The activation state and exhaustion of CAR T cells are influenced by changing conditions during the anticancer response, including the cancer burden and the cytokine milieu (107, 108). Achieving a balance between initial and sustained response, tumor control, and managing both acute and chronic toxicity is crucial for effective therapy.

The basic primary effector functions of artificial immune cells like CAR T, CAR NK, chimeric TcR-like receptor and transgenic TcR expressing cells, are antigen-specific cellular cytotoxicity, cytokine production and proliferation (20, 57, 109, 110).

In reality, the immune effector cells are composed of a multitude of different cell subsets with various effector functions. The main αβT cell subsets are CD4⁺ T helper cells and CD8⁺ cytotoxic T cells. Yet, the various T cell subsets have been subclassified by the T cell receptor profile, the gene expression signature including decisive transcription factors, secretion of cytokines and chemokines as well as their cytokine- and chemokine receptor profiles which determine their responsiveness, adaptability and their specific functions (111–113).

While chemokine receptors and integrins determine the homing and preferential residence of T cells in certain tissues (114) the multifaceted effector functions including cytotoxicity and immunomodulation mediated by both CD4⁺ and CD8⁺ cells lead to their subclassification (115).

CD4⁺ αβT cells are classified as Th1, Th2, Tregs, TFH, TH22, TH17 and TH9. Cytokines range from proinflammatory IFNγ and TNFα released by Th1 cells to counterbalancing immunosuppressive cytokines IL10 and TGF-ß by Tregs (111). The interplay is important not only to prevent harm by hyperinflammation (116) but also to restore functions in order to maintain a potent immune response and circumvent terminal exhaustion of T cells (117).

CD8⁺ αβT cells are classified as Tc1, Tc2, Tc9, Tc17 and Tc22 and share a similar spectrum of cytokines like CD4⁺ cells ranging from proinflammatory to immunosuppressive functions as of IL12 and IL4, respectively (115). In relation to cancer entity the number and composition of tumor infiltrating lymphocytes (TILs), the frequency of CD8⁺ T cells and their subsets vary substantially (115). The prognostic value of TILs (CD4⁺ and CD8⁺) depends on the cancer type and the pathological TMN staging (118, 119).

Importantly, the maturation state of T cells defines their proliferative and regenerative capacity and is identified by the expression of cytokine- and chemokine receptors as well as activation and exhaustion markers (111).

Culturing conditions, including the cell culture media (e.g., glucose level) and supplements such as sera and cytokines (e.g., IL2, IL7, IL15, IL21, IL18), as well as cytokine dosing, initiation of T-cell activation and proliferation, and the duration of expansion, significantly impact the cellular composition of CAR products. These factors determine the maturation state and stemness of CAR effector cells, which in turn affect their proliferative capacity and clinical performance (120). The duration of CAR T cell expansion is crucial for supply, cost, and performance. CD19CAR-T tisagenlecleucel (CTL019), manufactured using the T-Charge^TM platform, is known as YTB323. This platform reduces manufacturing time from 9-10 days to less than 2 days. YTB323 has demonstrated that shortened manufacturing time preserves the stemness of CAR T cells, measured as the frequency of T_N and T_SCM, and achieves the same tumor control as tisagenlecleucel at a 25-fold lower dose (121). Other strategies to induce stemness and enhance CAR T cell functionality include supplementing the culture media with inosine which induced profound metabolic reprogramming, from glycolysis to mitochondrial oxidative metabolism and the epigenome toward greater stemness. The same effect was induced by genetically modifying CAR T cells to overexpress adenosine deaminase (ADA-OE), an enzyme that metabolizes adenosine to inosine, thereby preventing inhibitory effects through the ATP, ADP, and AMP CD39/CD73 pathway (122). Additionally, overexpressing FOXO1, a transcription factor involved in regulating gluconeogenesis and glycogenolysis mediated by insulin, has been shown to enhance stemness, metabolic fitness, and CAR T cell performance (123).

Based on their immunomodulatory functions CD4⁺ and CD8⁺ cell subsets harmonize the immune response. Severe immediate or chronic overactivation of T cells can lead to detrimental effects and death observed in viral infections (124), autoimmunity, serotherapy during conditioning for allogeneic stem cell transplantation and CAR T cell therapy (125).

In all of the US-FDA approved CAR T cell products the extracellular recognition domain is either based on a murine scFv or a llama-derived VHH, hence contain non-human sequences, which may be identified by the immune system as foreign and induce the generation of CAR-rejecting antibodies (24) or T cell mediated rejection (126, 127). Both allorejection mechanisms can lead to the loss of the CAR T cell function or to the complete elimination of the CAR T cells (126). Since immunogenicity is impacted by molecular size, sequence dissimilarity and conformational structure, camelid VHH are in general considered to be less immunogenic. VHH are superior in chemical and physical properties including higher solubility, stability, smaller size (15kDa), they have a higher resemblance in sequence and conformational structure to human VHH compared to murine scFv (30kDa), and hence exhibit lower immunogenicity (128). Immunogenicity of non-human proteins can be reduced by humanization of murine scFvs and deimmunization of camelid VHH (24, 129). However, CAR T cells targeted to B-lineage associated antigens, such as CD19, CD20, CD22 and BCMA protect themselves from rejection as they deplete or interfere with the immune compartment that is responsible for the generation of anti-CAR antibodies (129).

CAR T cell products are defined by their original cell source and cell number. Unstimulated peripheral blood leukaphereses contain a median of 9.8x10⁹ total nucleated cells with 3.8x10⁹ total CD3⁺ cells (130). Thus, one of the most compromising aspects of CAR T cell generation is the misrepresentation of their entire primary heterogeneity in the whole T cell pool of an estimated 4x10¹¹ number of cells (131), 1^st due to the low number of cells used for manufacturing [1x10⁸] with a median transduction efficiency of 46% (132) which corresponds to 0.01% (one ten thousandth) of the T cell pool, and 2^nd the collection from only one body compartment, the peripheral blood (130, 132). The heterogeneity of T cells in the peripheral blood and tissues are defined by differential functions dependent on the expression of certain integrins and chemokine receptors explaining the migration and residence tendency of the T cells in which they organize a complex interplay of the immune system (114).

Through the manufacturing process, various factors significantly impact CAR T characteristics. This process involves the drastic non-physiological antigen-independent activation of the T cell receptor complex (CD3) usually via the CD3 epsilon chain, often in combination with the co-activation of the CD28 costimulatory receptor (20). Additionally, continuous exposure to high concentrations of interleukin 7 (IL7), a T cell growth factor and regulator of Th1 and Th2 cytokine production (133) and/or the Th1-type cytokines, e.g., IL2 (120), IL15 (120, 134), IL21 (120) and/or the co-incubation with irradiated feeder cells (135), along with transgene delivery and the constitutive signaling and potential tonic signaling of the CAR receptor (136), collectively transform CAR expressing cells into artificial immune cells distinct from physiologic T and NK cells. Despite this distinction, CAR T cells share foundational properties with naturally matured and regrowing immune cells (137). Consequently, CAR expressing cells show similar behaviors and effector functions like natural immune cells but also exert additional functions and lack characteristics of their natural counterparts and have distinct metabolic signatures dependent on the CAR architecture (22). Signaling from T cell receptors and CAR receptors are fundamentally different even though CAR receptors mimic the TcR function and recruit the same downstream signaling proteins and engage with the same signaling pathways (138). Site-specific integration of CAR receptors into the TRAC locus facilitates the transgene expression according to the complex gene regulation of the TcR (57). The physiological CAR transgene expression has been shown to improve the CAR performance. Novel non-viral knock-in strategies may lower cost, complexity and time of CAR manufacturing. The technical challenge was to improve the delivery of homology-directed repair (HDR) templates of single-stranded DNA encoding the transgene instead of double-stranded DNA to reduce the toxicities in the electroporated cells (139).

These revolutionary gene editing tools have inspired the idea of site-specific integration of a novel immune receptor design mimicking the TcR. By exchanging the variable alpha and beta chains of the TcR with the antibody variable chains (VL and VH) of an antibody, the sensitivity of this advanced artificial immune receptor (HLA-independent TcR) was substantially increased to outperform classic CAR receptor design, demonstrated for antigen low expressing cancer cell line variants (NALM6_CD19Low and MOLM13_CD70Low). Yet, the constitutive co-expression of CD80 and 4-1BBL was required to enhance persistence (110). Interestingly, after three decades of CAR research, the advancements of synthetic biology and gene engineering have revived the original idea of Zelig Eshhar’s T-body, the forefather of the modern scFv-based CAR receptor (140).

The availability and suitability of target antigens are the greatest challenges in CAR T cell therapy. Due to the high requirements for target antigens, such as high and stable expression at relevant levels and preferably overexpression of the targeted antigen on cancerous cells compared to physiological tissues (141), the selection of potential target antigens are limited (142). Furthermore, the main mode of action of CAR T cells is the recognition and signaling of the CAR receptor upon engagement with the strictly defined epitope on a target antigen (143). Thus homogenous antigen expression is important for successful CAR T cell therapy, even though low frequency of antigen heterogeneity can be addressed by so called bystander effects whereas heterogenous cancers and antigen loss require multitargeted CAR approaches (144, 145).

Bystander effects through the recruitment of immune effector cells, such as T cells, NK cells and macrophages in the tumor niche have been shown to significantly contribute to tumor control for instance in the CD19 CAR T cell trial ZUMA-1 (NCT02348216) (146) and in preclinical models (147) and shall be exploited using next generation CAR designs, engineered to inducibly express cytokines impacting on the cytokine milieu (148). These effects are relevant but shall not be overinterpreted to enable CAR T cells to successfully control antigen negative tumor lesions.

In clinical practice there are three main strategies (i-iii) pursued to balance potency and on-target-off-tumor toxicities in targeted immunotherapies utilizing antibodies, bispecific T cell engagers (BiTEs), antibody-drug conjugates (ADCs) and CAR T cells.

All strategies are characterized by the inability to target cancer specifically and thus require to appreciate the potency limit set by the on-target-off-tumor toxicities comparable to dose-limiting toxicities (149) and cumulative dose-limiting toxicities in conventional chemotherapies (150). Despite the highly antigen-specific targeting mechanism, antibodies and medicines derived thereof are targeted to surface expressed structures that are co-expressed in physiological tissues (107, 151). As a consequence, all tissues that express the targeted antigen are subject to on-target-off-tumor toxicities. The impact of this effect is defined by biological factors on the target expressing cells as well as the CAR expressing effector cells. On the target cells, these include target antigen expression level and the primary resistance or general susceptibility of the target cells to CAR mediated effects for which a first indication of tumor control with acceptable toxicity was documented using GD2- (152) and HER2-specific CAR T cells (153). On the CAR T cell side, the affinity of the recognition domain and the signaling components both define the potency and the toxicity mediated on the antigen positive cells (154).

The most successful strategy ⁱ⁾ in CAR T cell therapy is to accept the “complete” eradication of target antigen positive cells transiently and/or permanently. This has been observed in patients treated with CD19- and BCMA-targeted CAR T cells (23, 155, 156). Importantly, the level of the depletion of physiological antigen positive cells is a strong indicator of leukemia control in B-lineage acute lymphoblastic (107) and chronic lymphocytic leukemia (157). The function of the B-lineage compartment can be substituted by immunoglobulin replacement therapy and as such displays an exception in the human body since severe tissue damages or depletion of tissues in most organs are not compatible with life. However, the strategy to eliminate specific cell subsets in the body permanently is quite unique and is reserved for CAR T cell immunotherapy in B-lineage dependent oncology (4, 92) and it has been demonstrated to be beneficial in life-threatening autoimmune diseases, such as refractory systemic lupus erythematosus (SLE) (158).

The second strategy ⁽ⁱⁱ⁾ is to transiently accept severe toxicities and then terminate the CAR T cell function after a predefined period of time. This is especially important in AML because AML-associated immunotargets are co-expressed on vitally essential myeloid bone-marrow derived cells and progenitor cells (159). CAR T cell therapy including conditioning chemotherapy with fludarabine and cyclophosphamide but especially in AML is accompanied with severe myeloid toxicities leading to an immunocompromised state with an increased risk for life-threatening infectious complications (160).

The systematic preclinical evaluation of CD33-directed CAR T cell constructs identified the CD33-targeted lintuzumab-based 2^nd generation CAR construct (LIN-CD28-CD3Z) to be most efficacious (161) and led to the multicenter CD33-specifc CAR T cell trial (NCT03971799) for pediatric patients with relapsed and refractory AML. In this context CAR T cells are used for remission induction prior to subsequent allogeneic stem cell transplantation. Using CD33-targeted CAR T cells requires the profound elimination of the CAR T cell function post treatment via the conditioning regimen to save patients from experiencing ongoing myelosuppression, unless patients are in parallel to the CAR T cell application, transplanted with genetically modified hematopoietic stem cells with a gene knockout for the CAR-targeted antigen, such as CD33 (162) or CD45 (163) to exclude the recovering autologous hematopoietic system from the CAR-targeting mechanism.

In the third strategy ⁽ⁱⁱⁱ⁾ the on-target-off-tumor toxicity is tolerable and generally not life-threatening. This applies mainly to overexpressed target antigens like mesothelin (MSLN) (97), CEA cell adhesion molecule 5 (CEACAM5) (164), HER2 (153), GD2 (152), B7H3 (165), CD70 (166), IL13Ra2 (167) and others in solid cancers. The listed target antigens are expressed on a variety of different tissues or are upregulated in immune cells upon activation, such as CD70 (168, 169) and B7H3 (170). The toxicities experienced with CAR T cells for solid cancer treatment are generally tolerable at standard dose levels (152, 164) and even intracranially administered CAR T cells in brain cancers (glioblastoma multiforme) (167) are considered a legitimate approach. However, in some instances CAR T cells in solid cancer patients have been lethal due to on-target-off-tumor toxicities. In a patient with colorectal cancer, reportedly a high dose of HER2-specific CAR T cells (1x10¹⁰)CAR⁺ cells) induced a severe acute respiratory distress syndrome (ARDS) and subsequent cardiac arrest causing death within days (171).

One approach to overcome or limit CAR mediated on-target-off-tumor toxicities in physiological non-cancerous tissues is to reduce the affinity of the recognition domain (scFv, VHH) (172). In CD19 CAR T cells, the 40x lower recognition domain CAR induced significantly less toxicities including CRS and ICANS compared to FMC63-based CAR T cells in childhood BCP-ALL patients while demonstrating enhanced proliferative capacity and antitumor activity (108). However, reduced affinity of the recognition domain and reduced responsiveness based on the CAR architecture [4-1BB versus CD28 costimulatory domain] increases the risk for antigen low expressing tumors to evade the targeting mechanism whereas CAR persistence remains an independent discriminator of clinical success (4, 12, 108). Sparing toxicities by reducing the affinity has been demonstrated for ICAM-1 specific CAR T cells (micromolar affinity) in thyroid cancer (173), as well as in HER2- and EGFR-specific CAR T cells in various solid cancer models including ovarian and prostate (174). Genetic alteration of the CD3ζ chain signaling to reduce the number of immunoreceptor tyrosine-based activation motifs (ITAMs) alleviates exhaustion and can alter the antigen-density threshold of a CAR (175). Logic AND-gating circumvent on-target-off-tumor toxicity but in consequence lead to a sensitivity decrease of the CAR T cells since the CAR requires two independent signals for efficient CAR activation, e.g., the AND-gating of a dual-CAR construct [CEA-CD3ζ & MSLN-4-1BB] in which the CEA-CAR provides the CD3ζ signaling and the MSLN-CAR provides the 4-1BB costimulatory signaling (176).

Since allorejection limits the efficacy of CAR T cell therapies, strategies to reduce immunogenicity via de-immunization and humanization have become an integral part of CAR T design. Further, reduced immunogenicity is achieved by using fully human natural ligand- or receptor-based CAR T. By repurposing high affinity ligand-receptor interactions to target overexpressed ligands [NKG2DL via NKG2D] (177) and receptors [BCMA & TACI via APRIL] (8) on cancers have been explored in the preclinical setting and in clinical trials (178). However, the clinical success of ligand-based CAR T compared to conventional scFv-based CAR T design thus far has been disappointing in multiple myeloma (7).

Cytokine release syndrome (CRS), Immune effector cell associated neurotoxicity syndrome (ICANS) and Macrophage Activation Syndrome (MAS) are early onset toxicities, which can have fatal symptoms (179).

The acute CRS is a common systemic inflammatory response to the excessive secretion of cytokines in the CAR T cell activation and proliferation, which occurs in 77-93% of leukemia patients treated with CAR T cells. Upon CAR T cell activation, the increased cytokine concentrations, particularly of IL6, a pleiotropic and pro-inflammatory cytokine, co-activate macrophages and monocytes, which leads to a further secretion of cytokines, and can be diagnosed from the high levels of granulocyte-macrophage colony-stimulating factor (GMCSF) in the serum. CRS is symptomized by fever, rigors, hypoxia, nausea, and heart rhythm disorders, including tachycardia and arrhythmias. In addition to heart dysfunctions, multiorgan failure can cause life-threatening medical conditions. Detections at early stages can allow for specific anti-inflammatory therapy, for instance, using tocilizumab to block the IL6 receptors, which can minimize the adverse effects (3, 180).

In the beginning of CRS, the brain is protected from primary and secondary involvement of CRS by the blood-brain barrier. Hence, the migration of CAR T cells to the brain is slower compared to other compartments of the body. However, the secretion of IL1ß and IL6 triggers von Willebrand factors, a glycoprotein, which disrupts the highly selective semi permeability of the blood-brain barrier. This permits cytokines and cells to enter the central nervous system, which changes the cytokine concentration in the brain. The elevated cytokine levels in brain tissues have been linked to neurological complications (181).

20-70% of CAR T cell patients develop neurological symptoms, including initial manifestations of cognitive impairment, such as tremor, confusion, lethargy, mild expressive and receptive aphasia, stupor, apraxia and decreased attention, which can progress into severe ICANS, symptomized by agitation, status epilepticus, cerebral oedema and occasionally intracerebral hemorrhage (3, 182, 183).

In CD19-targeted CAR T cells, the primary on-target-off-tumor toxicity occurs in the B-lineage tissues due to the expression of CD19, a BCR co-receptor on B cells. However, due to low expressions of CD19 in neural cells, on-target-off-tumor toxicities may also occur in neural tissue in the hyperinflammatory state. Furthermore, there are associations between the serum elevations of cytokine IL15, IL3 and GM-CSF and the blood-brain barrier permeability and ICANS development and severity (181, 184). Nonetheless, the fundamental principles, responsible for the neural toxicities are not thoroughly comprehended and other factors are considered to influence the severity of ICANS symptoms.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are severe hyperinflammatory syndromes, associated with the pathological dysregulation of macrophage proliferation, which are mostly triggered by viral infections, especially Epstein-Barr virus infection, genetic lymphoid immune cell disorder, rheumatic autoimmune diseases, for instance the systemic-onset juvenile idiopathic arthritis, systemic lupus erythematosus and genetic mutations (3, 185–187). In addition, HLH and MAS can occur in the context of BiTE and CAR T cell immunotherapy (185). The excessive release of proinflammatory cytokines IL1, IL6, IL18 and tumor necrosis factor, triggered by the uncontrolled activation and proliferation of macrophages, leads to dysregulated immune activity and induces systemic inflammation (186). MAS is characterized by hyperinflammatory clinical features, including persistent fever, cytopenia, coagulopathy, liver insufficiency, hepatosplenomegaly, lymphadenopathy and multi-organ failure (3, 186). Laboratory diagnostic characteristics are a decrease in blood cell count due to hemophagocytosis, hyperferritinemia, abnormal coagulation profile, elevated triglycerides and high cytokine levels. The treatment of MAS usually involves immunosuppressive medications, such as corticosteroid and immunomodulatory drugs, for instance IL1 and IL6 signaling inhibitors (179, 181).

A significant constraint in the effectiveness of CAR T therapies, particularly in treating solid tumors, is the immunosuppressive nature of the tumor microenvironment (TME) (188). The TME is composed of different cell types, matrix proteins and secreted factors (189). Immunosuppressive cell types like myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs) produce anti-inflammatory cytokines, such as interleukin-10 (IL10), IL4, transforming growth factor-beta (TGF-β) that inhibit the CAR T function. These cytokines hinder CAR T function, impede proliferation, induce T-cell exhaustion, reducing their efficacy against tumor cells by limiting their cytotoxic and cytokine-producing abilities. Additionally, dense fibrogenic extracellular matrix, dysregulated tumor vasculature, and the hypoxic TME, characterized by limited nutrients and oxygen, restrict CAR T infiltration and function (188).

Efforts to enhance CAR T efficacy involve engineering these cells to resist the TME-induced exhaustion or express additional effector functions to promote persistence and functionality within the immunosuppressive TME.

One strategy to overcome exhaustion is to target T cell intrinsic pathways, such as programmed death-1 (PD-1) or TGF-β (190). Activation of the TGF-β receptor upon binding of TGF-β present in the TME upregulates CD70 expression and induce T-cell exhaustion with increased expression of the inhibitory receptors PD-1 and TIM-3 (191). Sustained PD-1 expression positively correlates with an exhausted phenotype and dysfunction of the T cells (192, 193). Pre-clinical data indicates that knocking out of PD-1 and/or TGF-β receptor II genes in CAR T cells with CRISPR/Cas9 technology improve the CAR T function against tumors expressing PD-L1 and/or TGF-β release (194, 195). Current clinical trials, evaluate anti-MUC1 CAR T cells with PD-1 knockout in advanced esophageal cancer (NCT03706326) and anti-EGFR CAR T cells with TGFBR2 knockout in EGFR-positive solid tumors (NCT04976218). Expression of dominant-negative PD-1 or TGF-β receptors, receptors lacking intracellular domains necessary for downstream signaling, also show promising improvement of the CAR T persistence and have been shown to be safe in a phase I clinical trial (196, 197). Combining CAR T therapy with checkpoint inhibitors with already approved immunotherapies, such as anti-PD-L1 or CAR T cells engineered to secrete PD-1 scFv or nanobodies, promote the anti-tumor activity (97, 198). Other approaches such as chimeric switch receptor-expressing CAR T cells like PD-1/CD28 or TGF-β/IL7 also seems to improve the persistence of CAR T (199, 200).

Another strategy to enhance the anti-cancer functions of CAR T cells and recruit resident immune cells by modulating the TME is to use CAR T cells designed to produce pro-inflammatory cytokines (201). Many different cytokines have been used to potentiate CAR T cell function, such as interleukin (IL) IL7 (202), IL15 (134), IL21 (203), IL12 (204) and IL18 (86). Anticancer activity is substantially induced by the highly potent pro-inflammatory cytokine IL12, which is naturally secreted by monocytic cells, e.g., dendritic cells and macrophages. IL12 enhances the cytotoxic capacity of both NK cells and T cells by inducing granzyme B and perforin production and interferon (IFN)-γ secretion (205, 206) and reduce the immunosuppressive effects of regulatory T cells (207). However, despite the tempting and promising enhanced anticancer function of pro-inflammatory cytokine support for CAR T cells and accessory immune effector cells, severe life-threatening toxicities limit their use and require the incorporation of advanced safety measures (208). The use of inducible promoters incorporating responsive elements of NFAT, AP-1, NFκB, or other transcription factors and their combinations facilitates activation-related gene expression (209–211). Alternatively, small molecule-regulated gene expression systems, such as the tetracycline-dependent Tet-On system, can improve the control of transgenes, including CAR expression (212) and cytokine secretion. While gene regulation can be complex and prone to leakage, site-specific integration of transgenes offers new opportunities to harness physiological gene regulation mechanisms (57, 110). This approach can reverse immune functions, such as integrating activating transgenes (e.g., CAR) into inhibitory loci like immune checkpoint receptors (213). Functional control of transgenes at the protein level is more reliable (214).