key: cord-0003911-q2w6kxb9
authors: Mata, Melinda; Gottschalk, Stephen
title: Engineering for Success: Approaches to Improve Chimeric Antigen Receptor T Cell Therapy for Solid Tumors
date: 2019-02-23
journal: Drugs
DOI: 10.1007/s40265-019-01071-7
sha: dbdb823e2dcebf23fa3525899b62e1cc82de9f98
doc_id: 3911
cord_uid: q2w6kxb9

While impressive clinical responses have been observed using chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies, limited clinical benefit has been observed using CAR T cells for a variety of solid tumors. Results of clinical studies have highlighted several obstacles which CAR T cells face in the context of solid tumors, including insufficient homing to tumor sites, lack of expansion and persistence, encountering a highly immunosuppressive tumor microenvironment, and heterogeneous antigen expression. In this review, we review clinical outcomes and discuss strategies to improve the antitumor activity of CAR T cells for solid tumors.

In the field of cancer immunotherapy, adoptive immunotherapy with T cells, genetically engineered to express chimeric antigen receptors (CARs), is a fast-growing approach to treat aggressive and recurring malignancies. CARs are engineered fusion proteins that couple the antigen recognition capability of an antibody with the effector function of an immune cell, thereby directing cell specificity towards a tumor cell [1] [2] [3] [4] . Unlike the T cell's conventional antigen recognition mechanism, CARs recognize antigens on the target cell surface in their unprocessed form and in a major histocompatibility complex (MHC)-independent manner ( Fig. 1) . In this way, CAR T cells are able to recognize antigenic epitopes that would normally not have been seen by T cells, and also circumvent immune evasion strategies by which tumors avoid MHC-restricted T cell recognition, such as decreased expression of MHC molecules and/or defects in antigen processing.

Remarkable clinical responses using CAR T cells for the treatment of CD19+ hematological malignancies have been observed [5] [6] [7] [8] [9] [10] [11] , leading to US Food and Drug Administration (FDA) approval of two CD19-CAR T cell products in 2017. In addition, remarkable, durable responses have been observed with the adoptive transfer of CAR T cells targeting B cell maturation antigen-positive (BCMA+) multiple myeloma [12] . However, clinical observations thus far for solid tumors and brain tumors have been disappointing, with only a handful of patients showing responses ( Table 1) . The significant variability in targeted antigen expression, CAR design, and heterogeneity of enrolled patients make it exceedingly difficult to compare outcomes. However, these clinical studies have highlighted key deficiencies of current CAR T cells and have provided the impetus for improvement and redesign in the research setting. In this review we summarize how the observed clinical results have shaped current approaches that are actively being investigated to overcome the hurdles for CAR T cell therapy for solid tumors.

CARs, originally termed T bodies and first developed by Zelig Eshhar [13, 14] , have now progressed to a more sophisticated single molecule that encompasses several facets of T cell activation and effector function. In its simplest form, a CAR molecule consists of an extracellular antigen recognition domain, a hinge, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen recognition domain most commonly consists of a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb) targeting a particular antigen but can also comprise ligands or peptides that bind to molecules expressed on the cell surface of tumors [15, 16] . Different hinges, long or small, have been evaluated, and studies indicate that the hinge is not only a structural component of the CAR but greatly influences its function [17] . Commonly used transmembrane domains include the transmembrane domain of CD28 or CD8ζ. Original CARs, called first-generation CARs, only contained the CD3ζ chain or the Fc receptor γ chain as an endodomain to activate T cell signaling upon antigen encounter. Results from 'first-in-human' clinical studies with firstgeneration CAR T cells for solid tumors showed safety but had rather disappointing antitumor responses and low persistence of infused T cells. Kershaw et al. [18] infused autologous CAR T cells targeting α-folate receptor (αFR-CAR) into patients with ovarian cancer. All 14 patients had progressive disease and αFR-CAR T cells only persisted 2-3 weeks, with a peak at 5 days [18] . In a separate study, firstgeneration CAR T cells targeting CD171 were infused into neuroblastoma patients, with one of six patients having a partial response that was seen at day 56 post T cell infusion but was not maintained [19] . Similarly, in vivo CAR T cell persistence was low and mainly seen within the first week post-infusion, with only one patient having detectable CAR T cells by polymerase chain reaction (PCR) at day 42.

Limited T cell persistence in these first trials is most likely due to several factors, including insufficient T cell activation from first-generation CARs that lack appropriate co-stimulation. Different co-stimulatory molecules such as CD28, 4-1BB, OX40, CD27, ICOS, or DAP12 have been added to enhance CAR T cell activation [1-4, 20, 21] . Depending on the number of added co-stimulatory domains, these CARs are referred to as second generation (one co-stimulatory endodomain) or third generation (two co-stimulatory endodomains).

The CD28 signaling domain, which is the canonical second signal for T cell activation, was therefore incorporated into CARs (Fig. 2) and subsequently has been shown to induce greater CAR T cell persistence in direct comparison to first-generation CARs in patients [22] . Several groups have also shown that activating 4-1BB signaling rather than CD28 signaling prevented exhaustion, improved T cell survival, and enhanced formation of central memory T cells (T CM ) in preclinical studies [23, 24] . To this end, a direct comparison between CD28-and 4-1BB-containing constructs is currently underway in the clinic using a CD19specific CAR (ClinicalTrials.gov identifier NCT01853631). The use of 4-1BB co-stimulation in CAR T cells for solid tumors is still under investigation, and only limited data are available with mesothelin-specific CAR T cells for pancreatic cancer (Table 1) , and with epidermal growth factor receptor (EGFR) variant III (EGFRvIII)-or interleukin (IL)-13 receptor subunit α2 (IL-13Rα2)-CAR T cells for high-grade glioma [25] [26] [27] . In addition, third-generation GD2-CAR T cells with a CD28.OX40.ζ endodomain have been evaluated in patients with neuroblastoma. While infusion of CAR T cells was safe, their antitumor activity was limited [28] . The activation of 4-1BB signaling by expressing 4-1BB ligand (4-1BBL) on the cell surface of T cells (co-stimulation in trans), as opposed to simultaneous signaling in typical CAR constructs, may also be a promising approach as it mimics physiological separation of T cell signaling and has also been shown to have potent antitumor properties [29, 30] .

In summary, studies have highlighted that no universal CAR construct for all malignancies exists, but rather each CAR must be optimized for the targeted antigen and each Physiological T-cell tumor setting. We are hopeful that current clinical studies with CAR T cells may shed more light on the best CAR construct and/or co-stimulatory domains in the context of various solid tumors. However, second-genetic modifications of CAR T cells or expressing CARs in less differentiated T cell subsets will also be important. These approaches are summarized in Fig. 3 , and are discussed in detail in Sects. 3, 3.4, 4, 5, and 7.

Robust antitumor activity is directly correlated with the expansion and persistence of infused cells, thereby making it a vital component of CAR T therapy [31] [32] [33] . In trials using CAR T cells directed towards CD4 for the treatment of HIV, CAR T cells were able to persist up to 9 years after infusion and modeling of acquired data showed a disappearance halflife of > 16 years [34] . In addition, CAR T cells targeting CD19+ chronic lymphocytic leukemia (CLL) were shown to persist up to 49 months, resulting in 4 of 14 complete responses and concurrent B cell aplasia [8] . These results demonstrate that it is possible for CAR T cells to expand and persist long-term in vivo. Adverse effects of CD19-CAR T cell therapies such as cytokine release syndrome (CRS) correlate with tumor burden [35] , suggesting that the presence of target antigen on malignant cells is critical for CAR T cell activation and expansion. However, whether antigenic stimulation by normal CD19+ B cells contributes to the long-term persistence of CD19-CAR T cells is difficult to ascertain. Antitumor T-cell Studies using CAR T cells in the context of solid tumors have unfortunately shown minimal expansion and persistence (Table 1) . To promote expansion and persistence after CAR T cell infusions in the solid tumor milieu, several approaches have been explored both preclinically and clinically.

Since T cells transition through various stages of differentiation that are characterized by a progressive loss of function, the differentiation status of CAR T cells is an important consideration for optimizing their expansion, persistence, and antitumor activity. Several preclinical studies have corroborated that the antitumor potential progressively decreases as T cells further differentiate from naïve (T N ) to the newly characterized memory stem cells (T SCM ) to T CM to effector memory (T EM ) and then effector T cells (T E ) [36] [37] [38] .

Most clinical trials thus far have relied on the infusion of genetically modified bulk T cells initially obtained from peripheral blood, potently activated with anti-CD3 and anti-CD28 mAbs, and then expanded ex vivo with IL-2. While effective in generating large numbers of T cells for adoptive therapy, these approaches often differentiate T cells further to the point of inferior persistence and heterogeneous function in vivo [39] . Preferentially selecting a defined CD8+ and CD4+ subset prior to infusion could lead to enhanced antitumor efficacy in the solid tumor setting as has been seen with CD19-CAR T cells for leukemia [11, 40, 41] . Indeed, one recent publication demonstrated that CAR CD4+ T cells have superior antitumor activity in preclinical high-grade glioma models, and that mixing these with CAR CD8+ T cells impaired their effector function [42] .

CARs have also been engrafted onto virus-specific T cells with the rationale that in vivo antigen stimulation and co-stimulation received after engagement of their native T cell receptor (TCR) will promote persistence of CAR T cells. However, there is an intricate interplay between the expressed CAR and native virus-specific TCR requiring detailed analysis [43] . These bi-specific T cells have accordingly shown increased expansion and persistence in neuroblastoma patients compared with T cells expressing the same CAR but lacking viral specificity [44] . A long-term followup of this study showed complete responses in three of 11 patients with bi-specific T cells persisting up to 96 weeks [45] . Clinical trials are currently underway for other solid tumors, including cytomegalovirus-specific human epidermal growth factor receptor 2 (HER2)-CAR T cells for glioblastoma (NCT01109095) and varicella zoster virus-specific GD2-CAR T cells for sarcoma (NCT01953900), and in part have been published [46] .

Transducing specific T cell subsets with CARs has also improved their effector function. For example, investigators have shown that expressing CARs with an inducible T cell co-stimulator (ICOS) endodomain in T helper (Th) 17 cells mediates potent antitumor activity [20, 21] . More recently, the same group of investigators has also shown that mixing CD4+ T cells expressing CARs with an ICOS co-stimulatory endodomain and CD8+ T cells expressing CARs with a 4-1BB co-stimulatory endodomain results in superior antitumor activity [21] . Careful analysis of ongoing CD19-CAR T cell therapy studies have also provided insight into which T cell subset is most effective. For example, for CLL antitumor activity of T cells expressing a CD19-CAR with a 4-1BB.ζ endodomain correlated with the presence of CD27+PD-1 (programmed death-1)-CD8+ CAR T cells expressing high levels of the IL-6 receptor in the T cell product [32] . The recent discovery that epigenetic programs are critical for T cell fate [47, 48] , and that epigenetic reprograming can halt T cell exhaustion undoubtedly has the potential to further increase the potency of CAR T cells. This is probably best highlighted by a case report in which lentiviral integration into the methylcytosine dioxygenase TET2 gene locus significantly enhanced CAR T cell function [49] .

In addition, various methods have been shown to 'halt' T cells in a less differentiated state to maximize therapeutic efficacy. The use of IL-7 and IL-15, as opposed to IL-2, during the ex vivo generation of CAR T cells can promote the frequency of CD8+CD45RA+CCR (C-C chemokine receptor) 7+ stem cell-like T cells that induce superior antitumor functionality [50, 51] . The IL-7/IL-15 cytokine combination for ex vivo generation of CD19-CAR T cells is currently being compared to IL-2 in the clinical setting (NCT02652910). Furthermore, the use of another γ c cytokine, IL-21, has been shown to prevent differentiation of genetically modified T cells and enhance antitumor activity compared to cells expanded in IL-2 [52, 53] .

Alternative approaches to prevent T cell differentiation include modulating metabolic or developmental pathways in T cells. For example, activation of the Wnt/β-catenin signaling pathway, involved in various stages of T cell development, can also delay T cell differentiation towards a more naïve phenotype with greater antitumor capabilities than memory T cells [54, 55] . Additional molecules that target metabolic pathways including the use the mammalian target of rapamycin (mTOR) inhibitor rapamycin [56] or AKT inhibitors [57] have been shown to promote the function of minimally differentiated memory cells. While these approaches have not yet been studied in the clinical setting for solid tumors, ex vivo expansion of CAR T cells that promote less differentiated cells may produce long-lasting antitumor effects after infusion. Lastly, a recent study indicates that successive cycles of chemotherapy significantly depletes T N subsets, arguing that it might be advisable to obtain a leukopheresis product at diagnosis in high-risk solid tumor patients for future CAR T cell production [58] .

Lymphopenia has been shown to augment T cell expansion and increase T cell responsiveness, possibly due to the destruction of regulatory cells or the increased levels of homeostatic cytokines such as IL-7. To this end, several studies have shown that lymphodepletion prior to adoptive T cell therapy, including CAR T cells, has improved both expansion and function of infused T cells [41, 59] . Lymphodepleting mAbs are one attractive strategy to replace chemotherapy. For example, infusion of a pair of rat CD45 mAbs, which have a short half-life in humans, prior to T cell transfer induced transient lymphodepletion, resulting in enhanced, albeit limited, expansion of adoptively transferred T cells [60] . Additionally, infusion of the humanized CD52 mAb alemtuzumab induces profound lymphodepletion; however, it was deemed unsuitable to aid T cell expansion since the infused T cells also express CD52 [61] . Preclinical studies have shown that T cells in which CD52 expression is silenced readily expand post-CD52 mAb infusion, and encouraging results from one early phase clinical study for CD19+ malignancies have been reported [62] .

The uses of common γ chain or pro-inflammatory cytokines that promote CAR T cell survival and augment the T cell antitumor immune response have been extensively explored. Exogenous cytokine administration of IL-2, IL-15, or IL-12 can be used to promote adoptive T cell expansion; however, there is concern regarding severe systemic toxicity [63] [64] [65] [66] . To ensure local production of cytokines within the tumor environment, transgenic expression of IL-15 has been shown to promote survival and expansion of gene-modified cells in preclinical models [67] [68] [69] [70] . Additionally, inducible IL-12 secretion after antigen encounter by CAR T cells has shown similar results in various preclinical murine models [71, 72] . However, 'first-in-human' studies of inducible IL-12 in tumor-infiltrating lymphocytes (TILs) still resulted in toxicities and prevented a dose escalation above 3 × 10 9 cells where transient clinical responses were seen in 63% of patients [73] . Recently, IL-12-secreting mucin (MUC)16specific CAR T cells showed more than five-fold lower expression of IL-12 compared with previously published reports, possibly due to either its construction in a tricistronic vector or location of the IL12 gene behind an internal ribosome entry site (IRES) [74] . A phase I clinical study to evaluate safety of these CAR T cells for solid tumors is currently underway (NCT02498912). Lastly, transgenic expression of IL-18 is actively being explored to enhance the effector function of CAR T cells in preclinical models [75] [76] [77] .

Cytokines that have minimal systemic toxicity, such as IL-7, have also been investigated. Expression of IL-7Rα in combination with IL-7 administration has been shown to enhance T cell expansion [78, 79] . In such an approach, T cells that were once unresponsive to IL-7 due to lack of the cytokine receptor are now able to receive survival and proliferation signals induced by IL-7. As an alternate strategy, investigators have expressed a constitutive active IL-7 receptor [80] . Other T cell homeostatic cytokines such as IL-21 have been tested in a preclinical setting and were shown to enhance CAR T cell efficacy [81] .

As infused T cells typically accumulate in the lung and shortly thereafter are also found in the liver and spleen, enhancing T cell homing to the tumor site is paramount to improve their antitumor activity and preventing potential adverse effects [18, 82] . Efficient T cell homing is a multistep process that involves adhesion molecules and chemokine gradients. Tumors, and cells within the surrounding microenvironment, can secrete low levels of chemokines to prevent the accumulation of tumor-specific T cells or secrete chemokines that preferentially attract pro-tumor Th2 cells, myeloid-derived suppressor cells, or regulatory T cells (Tregs) [83] . To overcome these obstacles, transgenic expression of CCR2 and CCR4 on CAR T cells resulted in enhanced trafficking of CAR T cells to tumor sites leading to increased tumor clearance in preclinical xenograft murine models [84] [85] [86] . Alternatively, a small peptide expressed on CAR T cells that blocks the negative effects of protein kinase A on TCR activation was shown to also upregulate C-X-C chemokine receptor (CXCR) 3 and the CD49d integrin expression on CAR T cells, leading to enhanced migratory and tumor infiltration properties [87] . Although these approaches have not been tested in the clinic with CAR T cells, an ongoing clinical study is currently in progress with autologous TILs genetically modified to express CXCR2 to evaluate trafficking of T cells in metastatic melanoma patients (NCT01740557).

Other approaches to enhance T cell homing to tumor sites include the use of oncolytic viruses that preferentially replicate in tumor cells and are genetically modified to secrete chemokines. For example, an oncolytic adenovirus armed to secrete IL-15 and the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) showed improved GD2-CAR T cell infiltration into tumors resulting in increased persistence and antitumor function in preclinical models [88] . In addition, simply injecting T cells into or in proximity to tumor sites avoids the need for T cells to home to tumor sites [89] . Recently, intracranial injections of IL-13Rα2-CAR T cells showed remarkable efficacy in one glioblastoma multiforme (GBM) patient with regression of intracranial and spinal metastases that lasted up to 7.5 months [26] . Separate phase I clinical studies to evaluate the intratumoral injection of HER2-CAR T cells in GBM patients (NCT02442297) and CAR T cells that recognize the epidermal growth factor receptor family in head and neck cancer patients (NCT01818323) are in progress. Lastly, regional delivery of mesothelin-CAR or fibroblast activation protein (FAP)-CAR T cells for treating pleural-based mesothelioma is also actively being explored (NCT02414269, NCT01722149).

Solid tumors show considerable variability in antigen expression to avoid immune recognition. Furthermore, despite the high complete response rates seen with CAR T cells for CD19+ leukemia, antigen loss variants (ALVs) have been described [90, 91] . Several strategies are being developed to target multiple antigens. These include designing CARs with two antigen recognition domains, expressing multiple CARs in one T cell, or infusion T cell products, which each express CARs with distinct specificity. These approaches have been explored in preclinical models for hematological malignancies [91] [92] [93] [94] and clinical testing for hematological malignancies targeting CD19 and CD22 or CD19 and CD20 is in progress (NCT03448393, NCT03241940, NCT03019055). For solid tumors, preclinical studies have shown that targeting HER2 and IL-13Rα2 with bispecific CAR T cells prevents the development of ALVs [95] . In addition, the expression of three CARs within a single T cell has been reported to overcome immune escape [96] . Another approach to prevent ALVs is to combine the infusion of CAR T cells with the injection of an oncolytic virus encoding a bispecific antibody, which recognizes an antigen that is distinct from the CAR target [97] .

Unlike hematological malignancies, solid tumors flourish in restrictive locations and create a harsh immunosuppressive microenvironment that prevents the function of CAR T cells and tumor-specific T cells in general. For example, tumor cells express ligands for immune checkpoints such as PD-1, Lag-3 (lymphocyte activation gene-3), Tim-3 (T cell immunoglobulin and mucin-domain containing-3), and TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), and produce enzymes such as indoleamine-2,3-dioxygenase (IDO) or arginase, which deplete the essential amino acids tryptophan and arginine, respectively [98, 99] . Other immunosuppressive molecules include adenosine, cytokines such as IL-4 and IL-10, and tumor growth factor (TGF)-β [100] [101] [102] [103] . Thus, once T cells have successfully homed to the tumor sites, they still have significant obstacles to overcome. These also include physical barriers, such as the surrounding tumor stroma, that prevent CAR T cells from encountering malignant cells. Ex vivo expanded T cells lack expression of the enzyme heparanase that is essential for the degradation of the extracellular matrix (ECM) surrounding solid tumors. Engineering CAR T cells to express heparanase is one strategy to overcome this limitation and resulted in improved antitumor activity for solid tumors in preclinical models [104] . Additionally, targeting components of the tumor stroma itself, such cancer-associated fibroblasts (CAFs), is a potential strategy not only to decrease collagen content of the ECM but also counteract the immunosuppressive tumor environment since CAFs secrete immunosuppressive factors, such as TGF-β [105] [106] [107] . Malignant and stromal cells can also attract immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), Tregs, or tumor-associated macrophages (TAMs), which can express cell surface molecules or secrete inhibitory cytokines that further dampen the function of antitumor T cells [108] . For example, tumor cells may express PD ligand 1 (PD-L1), which can inhibit the effector function of T cells. A combination strategy with PD-1-blocking antibodies and CAR T cells can therefore potentially augment antitumor effects against solid tumors [109, 110] . Additionally, several genetic modification strategies have been developed to render T cells resistant to this hostile environment, including transgenic expression of dominant negative receptors or signal converters, which convert T cell inhibitory signals into stimulatory signals. TGF-β is a widely used immune evasion strategy by tumors since it promotes tumor growth while drastically inhibiting tumor-specific cellular immunity [111] . The unfavorable effects of TGF-β can be counteracted by modifying T cells to express a dominant-negative TGF-β type II receptor (DNRII) [112, 113] . While this approach has not been evaluated for CAR T cells in a clinical study, this approach has been evaluated with cytotoxic T lymphocytes (CTLs) targeting Epstein-Barr virus (EBV)-positive lymphoma. Results of this clinical study suggest that DNRIImodified CTLs have improved antitumor activity comparison with unmodified CTLs [114] . While dominant-negative receptors only protect T cells from the immunosuppressive environment, 'signal converters' provide direct positive signals. For example, linking the extracellular domain of the TGF-β type II receptor (RII) to the endodomain of Toll-like receptor 4 (TLR4) results in a chimeric receptor that not only renders T cells resistant to TGF-β, but also induces T cell activation and expansion [115] . A similar approach has also been used to convert inhibitory functions of IL-4 into T cell stimulatory signals [116, 117] and is currently undergoing phase I testing for head and neck cancer (NCT01818323).

In addition to the discussed immune checkpoints that inhibit T cell function within the tumor microenvironment, competition for key nutrients including sugars, amino acids, and fatty acids and the hypoxic tumor microenvironment have also emerged as critical factors that restrict the antitumor activity of T cells [118, 119] . The interested reader is referred to recent review articles that discuss in detail immune-metabolism cells and how they can be manipulated for therapeutic intent [118] [119] [120] . Quiescent T cells rely on oxidative metabolism and generated adenosine triphosphate through oxidative phosphorylation (OXPHOS). Once activated, T cells rely on increased glycolysis resulting in lactate production. For CAR T cells, one study has highlighted that CARs with a CD28 co-stimulatory endodomain preferentially induce glycolytic metabolism, whereas CARs with a 4-1BB co-stimulatory endodomain induce mitochondrial biogenesis and OXPHOS [24] . Thus, the choice of the CAR endodomain significantly influences T cell metabolism post-stimulation. In addition, amino acids such as arginine and glutamine, which are often depleted within the tumor microenvironment, are critical for T cell proliferation [121] . Several strategies are currently being explored in preclinical models to improve the 'metabolic fitness' of adoptively transferred T cells. For example, the ex vivo loading of tumor-specific T cells with arginine resulted in improved antitumor activity. Other approaches include the use of metformin, which reduces hypoxia levels in tumors [122] .

Most antigens targeted thus far with CAR T cells are not exclusively expressed on tumors, and low levels of expression in normal tissue has resulted in unforeseen 'on target/off cancer toxicity' in the clinical setting. For example, first-generation carboxy-anhydrase-IX (CAIX)-CAR T cells recognized CAIX expression on non-malignant bile duct epithelial cells, resulting in liver toxicities [123] . This 'on target/off cancer' toxicity could be prevented by infusing a CAIX-specific mAb prior to the infusion of CAIX-CAR T cells [124] . One patient died after receiving 1 × 10 10 third-generation trastuzumab-based HER2-CAR T cells and IL-2 after lymphodepleting chemotherapy for the treatment of metastatic colon cancer. This severe adverse event was attributed to low-level HER2 expression on normal lung epithelia [125] . However, in a separate clinical study with second-generation FRP5-based HER2-CAR T cells, no dose-limiting toxicity was observed in 17 sarcoma patients that had received no lymphodepleting chemotherapy and up to 1 × 10 8 /m 2 T cells [126] . Thus, conditioning regimen, T cell dose, and/or CAR design may affect the incidence and severity of 'on target/ off cancer toxicity'. In this section we review (i) controlling CAR expression and affinity; (ii) engineering of T cells to limit their activation to tumor sites; and (iii) suicide genes as measures to prevent toxicities of CAR T cells.

While stable expression of CAR constructs on T cells is needed to have sustained antitumor responses, CAR expression from messenger RNA (mRNA) electroporation offers a unique opportunity to prevent and/or screen for off-target effects in solid tumors since gene expression is transient. For example, mRNA electroporated CAR T cells can serve as a first pass to test for toxic effects towards normal tissue that may also express the targeted antigen but at lower levels [25, 127, 128] . One patient who received multiple doses of mRNA-electroporated CAR T cells developed an IgE-mediated anaphylactic shock most likely triggered by the extracellular domain of the CAR that was derived from a murine mAb, highlighting another potential adverse effect of CAR T cell therapy [128] . Proof-of-concept studies have shown that T cells can be genetically engineered so that T cell recognition of one antigen expressed on tumor cells can induce expression of a CAR directed towards a second antigen (Fig. 4) , thereby relying on an 'antigen address' to initiate full CAR T cell activity towards tumors [129] . These include synthetic notch (synNotch) receptors, which consist of an antigen binding domain, a transmembrane domain, and a transcriptional activator. Once the receptor binds the target antigen, the transcriptional activator is cleaved and can then induce expression of a gene such as a CAR , which is under the control of the cleaved transcriptional activator [129] . As an additional method, modifying the affinity of antigen binding of the CAR could also potentially prevent the recognition of tumor antigen expressed at a low level on normal tissue, yet retain CAR activity against overexpressed antigens on tumor cells [130, 131] .

While tumor antigen discovery is actively being pursued, it might be impossible to discover single surface antigens that are uniquely expressed in solid tumors but not expressed on the cell surface of normal tissues. Tumors most likely express a unique pattern of antigens, which can be exploited using genetically modified T cells. For example, T cells have been engineered to express two CARs with different antigen specificity. One CAR provides antigen-specific ζ-activation, while the second CAR provides antigen-specific co-stimulation, thereby restricting full T cell activation to tumors, which express a 'unique tumor antigen address' (Fig. 4 ) [132] [133] [134] . For this approach to work, the two targeted antigens must not be present together in a single location within normal tissues; thus, antigen selection will be critical for this approach to work. Differential antigen expression can also be used to inhibit T cell signaling. In this strategy, T cells are engineered to expresses two CARs: (1) a typical CAR construct that can activate T cell signaling once a tumorassociated antigen has been recognized; and (2) a CAR construct with a scFv targeting an antigen that is expressed on normal tissue that contains endodomains with inhibitory signaling such as PD-1 or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) [135] . CAR T cells expressing these receptors have limited function at off-target sites but can still effectively maintain antitumor properties [135] . Inducible systems to regulate gene expression and/or turn on signaling pathways are an attractive approach to control activation and function of immune cells. Proof-of-concept studies have shown that inducible systems can also be utilized to control the expression and function of CAR T cells (Fig. 4) [129, 136] . Other approaches include controlling CAR expression or co-stimulation with a small molecule [137] [138] [139] . This control of CAR T cell function could potentially allow the clinician to infuse a relatively small amount of transgenic T cells and induce appropriate responses with the injection of a titratable drug. In such approaches, CAR expression and/or activation is only present if two molecular interactions occur, thereby making it more difficult for CAR T cells to initiate signaling, yet 'safer' against off-target effects towards normal tissue. For example, infused T cells, including CAR T cells, initially accumulate in the lung, before they migrate to tumor sites. Inducing CAR expression or co-stimulation once the bulk of CAR T cells have left the lung should limit potential toxicities to normal lung tissues.

If adverse events should arise, several approaches have been developed to completely turn off or ablate CAR T cells in vivo [140] . The first approach relies on the expression of an enzyme that activates a prodrug into a toxic compound. Clinical studies with T cells transduced with the herpes simplex virus thymidine kinase (HSV-tk) gene have shown that administration of the prodrug, ganciclovir, efficiently ablates HSV-tk-transduced T cells in vivo [141] . The second approach consists of genes that take advantage of dimerizing molecules and that can be specifically activated to induce T cell apoptosis. T cells that express an inducible caspase 9 gene (iC9) can be effectively ablated in preclinical models and in patients by administration of a small-molecule drug (chemical inducer of dimerization [CID], AP1903) [142, 143] . Additionally, repeated doses of CID were able to eliminate a residual percentage of repopulating cells that express low levels of iC9 [144] , indicating that repeated doses of CID to activate inducible genes are safe and functional. Other dimerizer systems take advantage of Fas [145] or other molecules in the apoptosis or necroptosis pathways, as reviewed elsewhere [146] . Lastly, expression of a cell surface antigen on engineered T cells such as truncated CD20 or EGFR allows the elimination of T cells with FDA-approved mAbs that induce complement activation and/or antibodydependent cellular cytotoxicity (ADCC) [147, 148] . Currently, several clinical trials are testing such an approach with CD19-CAR and CD123-CAR T cells using truncated EGFR (NCT02028455, NCT02706405, NCT01865617, NCT02146924, NCT02159495).

Although CAR T cells have shown impressive clinical benefit with lasting effects in CD19+ hematological malignancies, clinical application of CAR T cells for the treatment of solid tumors is still in its beginning phases, with only a handful of complete responses achieved. Hindered by the heterogeneity and complexity of the solid tumor microenvironment, current CAR T cells by themselves may not completely be able to eliminate established tumors. However, recent advances in understanding how CAR T cells function highlight that these cells can be further modified or combined with other treatment modalities to enhance their antitumor activity. Thus, we remain cautiously optimistic that additional genetic modifications of CAR T cells will enhance their activity against solid tumors in humans. Indeed, several genetic approaches that improve CAR T cell expansion, persistence, homing to tumor sites, and their ability to function in the hostile tumor microenvironment are in early phase clinical testing or set to be evaluated in clinical studies within the next 5 years.

Funding The authors were supported by US National Institutes of Health (NIH) 1R01CA173750 and 1R01NS106379-01A1, and Cancer Prevention and Research Institute of Texas (CPRIT) grant RP101335.

Melinda Mata has no conflict of interest; she is currently an employee of Immatics US, Inc. Stephen Gottschalk has patents and patent applications in the field of T cell therapy and gene therapy for cancer, is a consultant of ViraCyte, LLC, a member of the data safety monitoring board of Immatics US, Inc., and received research funding from Tessa Therapeutics LTD.

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Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation

CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients

4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors

Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells

Activity of mesothelin-specific chimeric antigen receptor T cells against pancreatic carcinoma metastases in a phase 1 trial

Regression of glioblastoma after chimeric antigen receptor T-cell therapy

A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

CAR T cells administered in combination with lymphodepletion and PD-1 inhibition to patients with neuroblastoma

T cell-encoded CD80 and 4-1BBL induce auto-and transcostimulation, resulting in potent tumor rejection

Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells

The pharmacology of T cell therapies

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia

Clinical pharmacology of tisagenlecleucel in B-cell acute lymphoblastic leukemia

Decade-long safety and function of retroviralmodified chimeric antigen receptor T cells

Kinetics and biomarkers of severe cytokine release syndrome after CD19 chimeric antigen receptor-modified T-cell therapy

Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates

Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy

A human memory T cell subset with stem cell-like properties

Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells

Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo

Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity

Chimeric antigen receptor signaling domains differentially regulate proliferation and native T cell receptor function in virus-specific T cells

Virus-specific T cells engineered to coexpress tumorspecific receptors: persistence and antitumor activity in individuals with neuroblastoma

Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma

HER2-specific chimeric antigen receptor-modified virus-specific T cells for progressive glioblastoma: a phase 1 dose-escalation trial

De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation

Epigenetic maintenance of acquired gene expression programs during memory CD8 T cell homeostasis

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells

Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors

IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy

IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response

Pharmacologic induction of CD8+ T cell memory: better living through chemistry

Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes

The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin

Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism

Naive T cell deficits at diagnosis and after chemotherapy impair cell therapy potential in pediatric cancers

Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes

Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients

The role of alemtuzumab in facilitating maintenance immunosuppression minimization following solid organ transplantation

Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells

IL-2: the first effective immunotherapy for human cancer

Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during firstin-human clinical trial of recombinant human interleukin-15 in patients with cancer

Phase II study of interleukin-12 for treatment of plateau phase multiple myeloma (E1A96): a trial of the Eastern Cooperative Oncology Group

IL-12 deaths: explanation and a puzzle

Efficacy of IL-2-versus IL-15-stimulated CD8 T cells in adoptive immunotherapy

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

Transgenic expression of IL15 improves antiglioma activity of IL13Ralpha2-CAR T cells but results in antigen loss variants

Eradication of neuroblastoma by T cells redirected with an optimized GD2-specific chimeric antigen receptor and interleukin-15

IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression

Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice

Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma

IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo

Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL-18

Engineered tumor-targeted T cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system

CAR T cells releasing IL-18 convert to T-Bet(high) FoxO1(low) effectors that exhibit augmented activity against advanced solid tumors

Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7

Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition

Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells

IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice

Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia

Chemokines in tumor progression and metastasis

Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b

Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor

T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model

CAR T cell therapy for solid tumors

Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and longlasting CD4-dependent tumor immunity

Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy

T cells expressing CD19/CD20 bispecific chimeric antigen receptors prevent antigen escape by malignant B cells

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies

Preclinical development of bivalent chimeric antigen receptors targeting both CD19 and CD22

Tandem CAR T cells targeting HER2 and IL13Ralpha2 mitigate tumor antigen escape

Trivalent CAR T-cells overcome interpatient antigenic variability in glioblastoma

Improving CART-cell therapy of solid tumors with oncolytic virus-driven production of a bispecific T-cell engager

Targeting the indoleamine 2,3-dioxygenase pathway in cancer

Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

Targeting adenosine for cancer immunotherapy

Targeting cancerderived adenosine: new therapeutic approaches

IL4 primes the dynamics of breast cancer progression via DUSP4 inhibition

Current status of interleukin-10 and regulatory T-cells in cancer

Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes

Antitumor effects of chimeric receptor engineered human T cells directed to tumor stroma

Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells

Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Reversing T-cell dysfunction and exhaustion in cancer

Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptortransduced human T cells in solid tumors

Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells

TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance

Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor

Dominant-negative TGF-beta receptor enhances PSMAtargeted human CAR T cell proliferation and augments prostate cancer eradication

Tumor-specific T-cells engineered to overcome tumor immune evasion induce clinical responses in patients with relapsed Hodgkin lymphoma

Transgenic expression of a novel immunosuppressive signal converter on T cells

Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4

CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation

Antitumor T-cell reconditioning: improving metabolic fitness for optimal cancer immunotherapy

Targeting T cell metabolism for improvement of cancer immunotherapy

Metabolic regulation of CAR T cell function by the hypoxic microenvironment in solid tumors

L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity

Efficacy of PD-1 blockade is potentiated by metformin-induced reduction of tumor hypoxia

Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience

Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity

Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2

Human epidermal growth factor receptor 2 (HER2)-specific chimeric antigen receptor-modified T Cells for the immunotherapy of HER2-positive sarcoma

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor

T cells expressing chimeric antigen receptors can cause anaphylaxis in humans

Precision tumor recognition by T cells with combinatorial antigen-sensing circuits

Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity

Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells

Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling

Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo

PD-1-and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

A Tet-on inducible system for controlling CD19-chimeric antigen receptor expression upon drug administration

Inducible activation of MyD88 and CD40 in CAR T cells results in controllable and potent antitumor activity in preclinical solid tumor models

Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40

Clinical impact of suicide gene therapy in allogeneic hematopoietic stem cell transplantation

HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft versus leukemia

An inducible caspase 9 safety switch for T-cell therapy

Inducible apoptosis as a safety switch for adoptive cell therapy

Serial activation of the inducible caspase 9 safety switch after human stem cell transplantation

A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease

Exploiting cell death pathways for inducible cell elimination to modulate graft-versus-hostdisease

An improved bicistronic CD20/tCD34 vector for efficient purification and in vivo depletion of gene-modified T cells for adoptive immunotherapy

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells

CD133-directed CAR T cells for advanced metastasis malignancies: a phase I trial. Oncoimmunology

Phase I escalating-dose trial of CAR-T therapy targeting CEA(+) metastatic colorectal cancers

The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity