Chimaeric antigen receptor (CAR) T-cell therapies, as one of the cancer immunotherapies, have heralded a new era of treating cancer. The accumulating data, especially about CAR-modified T cells against CD19 support that CAR T-cell therapy is a highly effective immune therapy for B-cell malignancies. Apart from CD19, there have been many trials of CAR T cells directed other tumour specific or associated antigens (TSAs/TAAs) in haematologic malignancies and solid tumours. This review will briefly summarize basic CAR structure, parts of reported TSAs/TAAs, results of the clinical trials of CAR T-cell therapies as well as two life-threatening side effects. Experiments in vivo or in vitro, ongoing clinical trials and the outlook for CAR T-cell therapies also be included. Our future efforts will focus on identification of more viable cancer targets and more strategies to make CAR T-cell therapy safer.

Introduction

Recently, cancer immunotherapies received a high degree of attention, which mainly contained the treatments for programmed death 1 (PD-1), programmed death ligand 1 (PD-1L), cytotoxic T lymphocytes-associated antigen 4 (CTLA-4) and chimaeric antigen receptors (CARs) [1]. Studies of (CAR)-specific T cells were viewed with exceptional interest for clinical development.

CAR T-cell therapy was a mode of adoptive T-cell therapy, which also contained tumour-infiltrating lymphocytes (TILs) and TCR engineered T cells (TCRT). However, TILs were reproducibly detectable only in a minority of cancers [2]. Also, the human leucocyte antigen (HLA) restricted nature of TCR recognition limited the application of TCRT to specific HLA repertoires. Fortunately, CAR T-cell therapy satisfied the need to explore new and efficacious adoptive T-cell therapy. CAR was a synthesized transmembrane protein, redirecting the target antigens expressed in tumour cells through genetic reprogramming. This gene transfer technology could efficiently introduce genes encoding CARs into immune effector cells [3]. Once transferred, engineered T cells provided specific antigens binding in a non-major histocompatibility complex (MHC) restricted manner, and were capable of recognizing tumour independently of HLA molecules. Therefore, compared with the TCRT therapy, the CARs might recognize a far greater range of potential cellular targets and could be applied to a broad range of patients irrespective of HLA phenotype [35]. These advantages, namely MHC-independent and tumour-specific were also carried by another approach, bi-specific T-cell engagers (BiTEs). BiTEs were a subclass of bi-specific antibodies (bsAbs). BiTEs did not need conventional MHC recognition when they induced T-cell activation through dual antigen binding. They were specific for CD3 on one arm and a tumour antigen on the second in order to bring T cells and malignant cells into close proximity [6]. However, Stone et al. [7] compared the in vitro sensitivity of these two strategies and found that CAR-expressing T cells were more sensitive than BiTE-treated T cells to low numbers of antigens per cell [7]. This indicated that CARs might be considered to be used in preference to BiTEs when epitope densities were low.

In decades, CAR T-cell therapy generated a great deal of enthusiasm in the field of cancer treatment. It made gratifying achievements for the treatment of haematologic malignancies like leukaemia [8] and lymphoma [9], as well as solid tumours such as neuroblastoma [1012] and glioblastomas [13,14].

In this review, we will summarize current achievements and challenges of the CAR T-cell therapy and focus on the strategies to maximize the potential of this therapy.

Structures, advantages and disadvantages of each generation of CAR

Over the last decades, a lot of attempts were made to construct the structures of CARs. Generally, CARs contained a targeting moiety, a transmembrane domain and an intracellular region. Specifically, a single-chain variable fragment (scFv) linked to a hinge region made up the targeting moiety, and the intracellular region was an immunoreceptor tyrosine-based activation motif (ITAM) which comprises either a region of CD3ζ chain or FcR receptor γ (FcεRIγ) [4,5].

Depending on the differences of intracellular signalling domains, CAR T cells were classified as first, second and third generation [15,16] (Figure 1). The first-generation CAR T cells just consisted of scFv and ITAM lacking co-stimulatory signalling. So, the activation and the proliferation of T cells were at a low level, leading to a short time of T-cell-killing and anti-tumour efficacy [17]. To address this limitation, the second-generation CAR T cells were designed, which expressed co-stimulatory molecules (CMs) in the intracellular domain. Concretely, they encompassed one CM such as CD28 and CD137 (4-1BB) [3,15]. The second generation showed strikingly enhanced expansion and persistence of T-cell activation, growth and survival [18]. In order to improve the efficacy, the third generation was developed based on the second generation. They had two CMs among CD28, CD27, 4-1BB and the others [3,16]. Inducted CMs into the CARs construction resulted in enhanced activation, proliferation and elevated survival of T cells so that the CAR T cells could exhibit more tumour cell-killing efficacy [16]. However, because of the presence of multiple intracellular signalling caused by the CMs of the second or third generation, an abundance of cytokines might be released and they would have resulted in cytokine storm, which was life threatening [19].

CAR T cells were classified into three generations based on intracellular signalling domains

Figure 1
CAR T cells were classified into three generations based on intracellular signalling domains

First-generation CARs contained only one signalling domain. To provide the needed co-stimulatory receptors, CD28 or 4-1BB were integrated into the second-generation CARs. Third-generation CARs had two co-stimulatory domains, typically included both CD28 and 4-1BB or CD134 (OX40). Besides this, the concept of the ‘TRUCK’ was raised. They were produced through the introduction of additional genes, including those encoding T-cell–co-stimulatory ligands (4-1BBL) or pro-inflammatory cytokines (interleukin (IL)-12).

Figure 1
CAR T cells were classified into three generations based on intracellular signalling domains

First-generation CARs contained only one signalling domain. To provide the needed co-stimulatory receptors, CD28 or 4-1BB were integrated into the second-generation CARs. Third-generation CARs had two co-stimulatory domains, typically included both CD28 and 4-1BB or CD134 (OX40). Besides this, the concept of the ‘TRUCK’ was raised. They were produced through the introduction of additional genes, including those encoding T-cell–co-stimulatory ligands (4-1BBL) or pro-inflammatory cytokines (interleukin (IL)-12).

Besides, the concept of the fourth-generation CAR-modified T cells, which was also known as ‘TRUCK’ T cells, was raised by some studies [20]. The fourth-generation CAR T cells with additional genetic modification were able to express proliferative T-cell–co-stimulatory ligands (4-1BBL) or pro-inflammatory cytokines (IL-12) (Figure 1) [3]. Once recognizing the TSAs/TAAs on the tumour cells, the fourth-generation CAR T cells released a large number of perforins, granzymes and tumour necrosis factors (TNFs), which eventually led to apoptosis of tumour cells. Compared with the first three generations, the ‘TRUCK’ T cells had more advantages on affecting local suppressive cells and were enable to cause more anti-tumour destruction [21].

TSAs/TAAs for CAR T-cell therapy

A multitude of CARs targeting an array of TSAs/TAAs have been reported for their remarkable anti-tumour effect in vitro or in vivo, including targeting cell surface tumour antigens in haematologic malignancies and solid tumours [15]. Generally, antigens recognized by CARs needed to be expressed on the surface of the tumour, making it an important disadvantage. However, interestingly, certain article showed that intracellular tumour antigens might also be recognized using the cell receptor-mimic antibody (TCRm) CAR, which derived from the ESK1 TCRm monoclonal antibody (mAb) [22]. Moreover, tumour angiogenesis was an ideal choice of the targets for CAR T-cell therapy as well [23,24]. All these possibilities made CAR T-cell therapy a powerful tool for cancer treatments.

Cell surface tumour antigens in haematologic malignancies

CD19

In clinical trials, CD19 was most widely used as a target tumour antigen of haematological cancers (Table 1). CD19 was considered as an ideal target for B-cell malignancies because of its high and uniform expression on B cells [25]. In 2014, Kochenderfer et al. [9] published the first report on successful treatment of diffuse large B-cell lymphoma (DLBCL), and demonstrated the feasibility and validity of anti-CD19 CAR T cells treating chemotherapy-refractory B-cell malignancies. The present study concluded 15 patients, eight achieved complete remissions (CRs). The result indicated anti-CD19 CAR T-cell therapy was a potentially powerful new treatment for B-cell malignancies. In the same year, another clinical trial showed that CR was achieved in 27 patients among a total of 30 children and adults with relapsed or refractory acute lymphoblastic leukaemia (ALL) [8]. These anti-CD19 CAR T-cell therapy overcame limitations of conventional therapies and induced remission in patients with refractory disease and the effectiveness provided strong support for further development of this approach.

Table 1

Published results from clinical trials of CAR T cells targeting CD19 and CD20 in haematologic malignancies

Antigens References Diseases Responses to CAR T cells Main side effects 
CD19 [64Two FL Two NR None 
CD19 [18Six NHL Two SD, four PD None 
CD19 [75Eight CLL, one ALL Two SD, one reduction in lymphadenopathy, three no objective response, one B-cell aplasia, one PD, one NE Fever 
CD19 [76Three CLL Two CR, one PR TLS, SIRS, B-cell aplasia 
CD19 [19Three FL, four CLL, one SMZL One CR, five PR, one PD, one NE SIRS, B-cell aplasia 
CD19 [54Two ALL Two CR SIRS, CNS toxicity 
CD19 [77Four ALL, four CLL Two CCR, one CR, one PR, one SD, three PD None 
CD19 [78Four CLL, Four MCL, two DLBCL One CR, one PR, six SD, two PD TLS, SIRS, fever 
CD19 [5316 ALL 14 CR SIRS, neurotoxicity 
CD19 [9Nine DLBCL, four CLL, two indolent lymphomas Eight CR, four PR, one SD, two NE SIRS, CNS toxicity 
CD19 [79One DLBCL, 20 ALL 14 CR, three SD, four PD SIRS 
CD19 [830 relapsed and refractory ALL 27 CR, three NR SIRS 
CD19 [80Nine relapsed and refractory ALL Two MRD, two CR, three PD, one CNS1, one haematological improvement and reduction in blast counts of bone marrow CRS, neurotoxicity 
CD19 [8114 relapsed and refractory CLL Four CR, four PR, six NR CRS, B-cell aplasia 
CD19 [82One MM One CR B-cell aplasia 
CD20 [83Seven FL Two CR, one PR, four SD Fever 
CD20 [64Two DLBCL Two in remission continually after autologous haematopoietic stem cell transplantation Cytopenias 
CD20 [27Two MCL, one FL Two without evaluable disease remained free of progression, one PR Fever, cytopenias 
CD20 [84Seven DLBCL One CR, three PR, one SD, one PD, one NE TLS, CRS 
Antigens References Diseases Responses to CAR T cells Main side effects 
CD19 [64Two FL Two NR None 
CD19 [18Six NHL Two SD, four PD None 
CD19 [75Eight CLL, one ALL Two SD, one reduction in lymphadenopathy, three no objective response, one B-cell aplasia, one PD, one NE Fever 
CD19 [76Three CLL Two CR, one PR TLS, SIRS, B-cell aplasia 
CD19 [19Three FL, four CLL, one SMZL One CR, five PR, one PD, one NE SIRS, B-cell aplasia 
CD19 [54Two ALL Two CR SIRS, CNS toxicity 
CD19 [77Four ALL, four CLL Two CCR, one CR, one PR, one SD, three PD None 
CD19 [78Four CLL, Four MCL, two DLBCL One CR, one PR, six SD, two PD TLS, SIRS, fever 
CD19 [5316 ALL 14 CR SIRS, neurotoxicity 
CD19 [9Nine DLBCL, four CLL, two indolent lymphomas Eight CR, four PR, one SD, two NE SIRS, CNS toxicity 
CD19 [79One DLBCL, 20 ALL 14 CR, three SD, four PD SIRS 
CD19 [830 relapsed and refractory ALL 27 CR, three NR SIRS 
CD19 [80Nine relapsed and refractory ALL Two MRD, two CR, three PD, one CNS1, one haematological improvement and reduction in blast counts of bone marrow CRS, neurotoxicity 
CD19 [8114 relapsed and refractory CLL Four CR, four PR, six NR CRS, B-cell aplasia 
CD19 [82One MM One CR B-cell aplasia 
CD20 [83Seven FL Two CR, one PR, four SD Fever 
CD20 [64Two DLBCL Two in remission continually after autologous haematopoietic stem cell transplantation Cytopenias 
CD20 [27Two MCL, one FL Two without evaluable disease remained free of progression, one PR Fever, cytopenias 
CD20 [84Seven DLBCL One CR, three PR, one SD, one PD, one NE TLS, CRS 

CCR, continuous complete response; CLL, chronic lymphocytic leukaemia; CNS, central nervous system; CNS1, no detectable leukaemia in the cerebrospinal fluid; CR, complete response; CRS, cytokine release syndrome; FL, follicular lymphoma; MCL, mantle cell lymphoma; MM, multiple myeloma; MRD: minimal residual disease; NE, not evaluable; NHL, non-Hodgkin’s lymphoma; NR, no responses; PD, progressive disease; PR, partial response; SD, stable disease; SIRS, systemic inflammatory response syndrome; SMZL, splenic marginal zone lymphoma; TLS, lysis syndrome.

CD20

CD20 (Table 1) was an activated glycosylated phosphoprotein expressed on the surface of B-lymphocytes [26]. Till et al. [27] conducted a pilot clinical trial aiming to test the effect of a third-generation CD20-specific CAR on patients with relapsed indolent B-cell and mantle cell lymphomas. The treatment was well tolerated and the clinical results of this therapy were promising with one patient having an objective partial response and two remaining free of progression for 12 and 24 months. This year, Watanabe et al. [26] concluded a research that anti-CD20 CAR T-cell therapy might also be an applicable option for the treatment of CD20-positive lymphoid malignancies. What’s more, the report found out a threshold of the antigen density. The threshold was sufficient for practical effectiveness, meanwhile, it might not result in adverse effects. Thus, antigen density seemed an ideal point for further investigation to reduce side effects.

CD30

CD30 was a member of the TNF receptor superfamily [28]. The malignant cells in a broad variety of Hodgkin’s lymphoma (HL) and NHL selectively express CD30, which was considered as an alternative target antigen [29,30]. A phase I dose escalation study summed up that eight of nine patients with relapsed/refractory CD30 + HL or NHL treated by CAR CD30-T cells had either relapsed or progressed, showing objective anti-tumour responses [31]. However, lymphocytes and haematopoietic stem and progenitor cells (HSPCs) also expressed CD30 after activation. As for this concern, a recent research provided evidence that therapy with anti-CD30 CAR T cells derived by HRS3scFv displayed a superior therapeutic index in the treatment of CD30 + malignancies without attacking healthy activated lymphocytes and HSPCs [30]. All of these demonstrated that anti-CD30 CAR T-cell therapy could be alternative therapeutic strategy for patients with resistant/relapsed lymphomas.

CD33

CD33 was a myeloid differentiation antigen unexpressed on pluripotent haematopoietic stem cells or inside the haematopoietic system. It could be displayed on some normal B cells, activated T cells and natural killer (NK) cells, validated as an AML target [32]. A second-generation CD33-specific CAR was generated and was proved to be effective for acute myeloid leukaemia. In the present study, leukaemia cell lines and primary tumour cells were efficiently killed in vitro by CAR T cells. Furthermore, the number of tumour cells was lower in mice treated with anti-CD33 CAR T cells than in control-treated mice. It showed that the anti-CD33 CAR T cells were also effective in vivo [33]. Therefore, anti-CD33 CAR T-cell treatment was highly effective in preventing AML development.

CD123

CD123 was an attractive surface target highly expressed in leukaemic stem cells and leukaemic blasts but lowly expressed in normal HSPCs [34]. Mardiros et al. [35] found that their CD123 CAR T cells exhibited potent effector activity in vitro as well as anti-leukaemic activity in vivo against a xenogeneic model of disseminated AML. Another animal experiment supported that CAR T-cell therapy was a viable therapy for AML by targeting of CD123 via CAR-engineered T cells [36]. Therefore, all of these results suggested that CD123 CAR T-cell therapy was a promising immunotherapy and CD123 CAR T cells were potent candidates for future treatment of AML.

Cell surface tumour antigens in solid tumours

Prostate specific membrane antigen (PSMA)

PSMA was a 750-amino acid type II membrane-bound glycoprotein and abundantly expressed on the endothelium of many solid tumours, dramatically in prostate cancer [37]. A preclinical model was proposed that a second-generation anti-hPSMA CAR T cells exhibited evident and specific anti-tumour activity against a prostate tumour model, both in vitro and in vivo [38]. An experiment developed a second-generation anti-PSMA CAR T cells for improving the efficacy of first generation. The results described that the second-generation secreted more cytokines and proliferated more vigorously in vitro than the first generation. What’s more, the second-generation appeared to be with higher potency on suppressing prostate tumour growth in animal models [39]. Both results provided the basis for advancing such approach towards clinical application.

Epidermal growth factor receptor variant type III (EGFRvIII)

EGFRvIII, a neo-antigen expressed in approximately 30% of glioblastomas and was correlated with poor prognosis [40]. According to Miao et al. [13], they established intracranial D-270 MG tumours, and showed that EGFRvIII CAR T cells had the capacity to suppress tumour growth and enhance survival of mice. Another report demonstrated that mice with glioma were successfully treated with a third-generation EGFRvIII CAR T cells. Significantly, the results endorsed clinical translation of this CAR in patients with brain tumours expressing EGFRvIII. [14]. Because of its highly specific and promising therapeutic efficiency, the results of clinical trials using EGFRvIII CAR T cells to treat glioblastoma attracted extensive attention.

Disialoganglioside (GD2)

GD2 was overexpressed among paediatric and adult solid tumours, such as neuroblastoma, retinoblastoma, glioma, Ewing’s family of tumours and many other solid tumours [41]. A study tested T cells carrying the anti-GD2 CAR. The results displayed anti-cancer killing activity both in neuroblastoma cells in vitro and in vivo xenograft studies. More importantly, clinical testing of the approach was warranted in neuroblastoma and other GD2-positive malignancies due to the promising results of the study [10]. What’s more, two trails also support the anti-tumour effects of the CAR T cells specific for the GD2. One of the trails reported that four patients with neuroblastoma had evidence of tumour necrosis, including a sustained complete remission. And there were no adverse events of the therapy seen in the total 11 subjects followed for up to 24 months. The second one demonstrated three patients achieving complete remission among 11 patients with active disease of neuroblastoma and observed long-term low-level presence of CAR expressing T cells was associated with longer survival [11,12]. GD2-targeting CARs, therefore, afforded us an alternative method for treatments of neuroblastoma.

Intracellular tumour antigens

Wilms tumour 1 (WT1)

WT1 was overexpressed in many cancers, including haematologic malignancies, like acute and chronic leukaemias and numerous solid tumours. A study designed WT1 28z CAR T cell, the first one against a human intracellular protein, WT1. WT1 28z T cells were specific for the WT1-HLA-A*02:01 complex and the outcome provided the proof-of-concept that CAR T cells could not only target the protein expressed on the cell surface of the tumour, but also target at intracellular antigens [22].

CAR T cells targeting tumour angiogenesis

Vascular endothelial growth factor receptor 2 (VEGFR-2)

Tumour angiogenesis could also be a target for CAR T cells, except TSAs/TAAs. Some approaches, using VEGFR-2 CAR, aimed at targeting the tumour vasculature rather than the tumour cells because VEGFR-2 was overexpressed in tumour vasculature and was related to tumour progression and metastasis [42]. Chinnasamy et al. [23] developed a method to target tumour vasculature and the result was that the growth of five different types of established, vascularized syngeneic tumours was significantly inhibited by VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 and the survival of mice was prolonged. Their late-stage study further displayed that co-administration of anti-VEGFR2 CAR T cells along with cells expressing a tumour-specific TCR could lead to a synergistic anti-tumour effect. Meanwhile, tumour-free survival of mice with established cancers was prolonged. All of these approaches targeting tumour angiogenesis opened new possibilities for the treatment of a wide variety of cancer types [24].

Other antigens in haematologic malignancies and in solid tumours were also well studied in vitro or in vivo, such as CD138 in MM [43], natural killer group 2 member D (NKG2D) in leukaemia and carcinoembryonic antigen (CEA) in colorectal cancer (Tables 2 and 3) [44].

Table 2

CAR T-cell therapies targeting other antigens in haematologic malignancies

Antigens Diseases In vitro, in vivo, in preclinical or in clinical trials NCT ID or references 
TRAIL receptor 1 Lymphoma In vitro [85
Kappa Lymphoma Clinical trial NCT00881920 
CD22 FL, ALL, NHL Clinical trial NCT02315612 
HA-1 H Leukaemia In vitro [86
NKG2D Leukaemia Clinical trial NCT02203825 
FAP B-cell CLL Clinical trial NCT01722149 
ROR1 CLL Clinical trial NCT02194374 
CD138 MM In vitro [43
 MM Clinical trial NCT01886976 
NY-ESO-1 MM In vivo [87
Lewis Y MM Clinical trial NCT01716364 
Antigens Diseases In vitro, in vivo, in preclinical or in clinical trials NCT ID or references 
TRAIL receptor 1 Lymphoma In vitro [85
Kappa Lymphoma Clinical trial NCT00881920 
CD22 FL, ALL, NHL Clinical trial NCT02315612 
HA-1 H Leukaemia In vitro [86
NKG2D Leukaemia Clinical trial NCT02203825 
FAP B-cell CLL Clinical trial NCT01722149 
ROR1 CLL Clinical trial NCT02194374 
CD138 MM In vitro [43
 MM Clinical trial NCT01886976 
NY-ESO-1 MM In vivo [87
Lewis Y MM Clinical trial NCT01716364 

FAP, fibroblast activation protein; NY-ESO-1, New York-oesophageal-1; ROR1, receptor tyrosine kinase-like orphan receptor 1; TRAIL receptor 1, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1.

Table 3

CAR T-cell therapies targeting other antigens in solid tumours

Antigens Diseases In vitro, in vivo, in preclinical or in clinical trials NCT ID or references 
HER2 Osteosarcoma In vitro [88
 Breast cancer In vitro [89
 Sarcoma Clinical trial NCT00902044 
 Metastatic cancer Clinical trial NCT00924287 
 Glioblastoma Clinical trial NCT01109095 
 Solid tumours Clinical trial NCT01935843 
CEA Colorectal cancer In vivo [44
 Colorectal cancer Clinical trial NCT00673322 
 Breast cancer Clinical trial NCT00673829 
 Liver metastases Clinical trial NCT01373047 
 Metastatic cancers Clinical trial NCT01723306 
CSPG4 Melanoma, breast carcinoma In vivo [90
EphA2 Glioblastoma In vivo [91
FR Ovarian cancer In vivo [92
IL-11Rα Osteosarcoma In vivo [93
IL-13Rα2 Glioblastoma Preclinical trial [94
 Malignant blioma Clinical trial NCT02208362 
 Refractory brain neoplasm   
 Recurrent brain neoplasm   
IL-13R Glioma Preclinical trial [95
CD171 Neuroblastoma Clinical trial NCT02311621 
EGFR Advanced EGFR-positive solid tumours Clinical trial NCT01869166 
 Advanced glioma Clinical trial NCT02331693 
Antigens Diseases In vitro, in vivo, in preclinical or in clinical trials NCT ID or references 
HER2 Osteosarcoma In vitro [88
 Breast cancer In vitro [89
 Sarcoma Clinical trial NCT00902044 
 Metastatic cancer Clinical trial NCT00924287 
 Glioblastoma Clinical trial NCT01109095 
 Solid tumours Clinical trial NCT01935843 
CEA Colorectal cancer In vivo [44
 Colorectal cancer Clinical trial NCT00673322 
 Breast cancer Clinical trial NCT00673829 
 Liver metastases Clinical trial NCT01373047 
 Metastatic cancers Clinical trial NCT01723306 
CSPG4 Melanoma, breast carcinoma In vivo [90
EphA2 Glioblastoma In vivo [91
FR Ovarian cancer In vivo [92
IL-11Rα Osteosarcoma In vivo [93
IL-13Rα2 Glioblastoma Preclinical trial [94
 Malignant blioma Clinical trial NCT02208362 
 Refractory brain neoplasm   
 Recurrent brain neoplasm   
IL-13R Glioma Preclinical trial [95
CD171 Neuroblastoma Clinical trial NCT02311621 
EGFR Advanced EGFR-positive solid tumours Clinical trial NCT01869166 
 Advanced glioma Clinical trial NCT02331693 

CSPG-4, chondroitin sulfate proteoglycan-4; EGFR, epidermal growth factor receptor; EphA2, Eph tyrosine kinase receptor A2; FR, folate receptor; HER2, human epidermal growth factor receptor 2.

Two serious side effects of CAR T-cell therapy and corresponding strategies

Although positive anti-tumour effects of the CAR T-cell therapies were mentioned above, some therapies targeting other TSAs/TAAs showed obvious toxicities rather than clinical benefits (Figure 2). A case of ‘on-target, off-tumour’ toxicity was reported that one patient receiving a third generation of erbB-2-targeted CARs died on 5th day after therapy. The patient experienced respiratory distress within 15 min after cell infusion. Investigators speculated that the reason was that lung epithelial cells also expressed low level of erbB-2 recognized and damaged by CAR T cells with released mounts of cytokines [45]. In order to overcome this toxicity, it is crucial to find more specific antigens expressed on tumour cells. However, majority of the antigens also keep low density on normal tissues [46]. In considering the difficulty to find truely TSAs or TAAs, Kloss et al. [47] presented a ‘dual-targeting’ strategy. The approach transduced T cells with a CAR that provided suboptimal activation and a chimaeric co-stimulatory receptor (CCR) (Figure 3). The suboptimal activation was upon binding of one antigen whereas the CCR was used to recognize a second antigen. T cells engineered in this manner would recognize the tumour, whereas tissues expressing either antigen alone would not activate them [47]. The efficiency of T-cell activation is ineffective in the absence of simultaneous CCR recognition of the second antigen. Similarly, T cells do not react against issues only expressing the CCR because T cells would not be activated, lacking the CAR. Another alternative strategy is to employ both activating and inhibitory CAR (iCAR) that operated as logic gates (Figure 4). The iCAR delivered a dominant inhibitory signal such as PD-1 or CTLA-4 to achieve antigen-specific suppression of T-cell cytotoxicity, and the activating CAR was capable of full T-cell activation. Tumour cells expressing only the activating ligand could operate T-cell function, whereas normal cells expressing both antigens could inhibit T-cell function [48]. These approaches thereby could improve tumour selectivity.

Strategies that could solve the problems including ‘on-target, off-tumour’ toxicity, ‘CRS’, ‘gene mutation’ and ‘autoimmune disorders’ and that might generate better CAR T products were taken into account

Normal cells that expressed only one antigen did not fully activate T cells

Figure 3
Normal cells that expressed only one antigen did not fully activate T cells

T cells expressing both CCR and suboptimal activation receptor were sufficiently activated by A+B+ target cells.

Figure 3
Normal cells that expressed only one antigen did not fully activate T cells

T cells expressing both CCR and suboptimal activation receptor were sufficiently activated by A+B+ target cells.

Normal cells could inhibit T-cell function when a CAR capable of full T-cell activation was co-expressed with an iCAR delivering PD-1 or CTLA-4

Figure 4
Normal cells could inhibit T-cell function when a CAR capable of full T-cell activation was co-expressed with an iCAR delivering PD-1 or CTLA-4

However, tumour cells fully activated T cells in the absence of the target for inhibitory signal.

Figure 4
Normal cells could inhibit T-cell function when a CAR capable of full T-cell activation was co-expressed with an iCAR delivering PD-1 or CTLA-4

However, tumour cells fully activated T cells in the absence of the target for inhibitory signal.

Additionally, antigen density seemed an ideal point regarding reduction of side effects. Watanabe et al. [26] found out a threshold for antigen selection, below which might not result in adverse effects, whereas that displayed practical effectiveness above the threshold. On the other hand, the possibility of generating two CARs that differ in affinity was raised. CAR T cells in the present study were described to have the ability to distinguish malignant from normal cells based on antigen density. In the present study, EGFR-specific CAR T cells bearing low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR. However, they displayed diminished effector function when the density of EGFR decreased. In contrast, activation of T cells with high-affinity cetuximab-CAR showed no change [46]. Similar findings were reported including EGFRvIII-specific CAR and ROR1-specific CAR [49,50]. These methods of tuning sensitivity of CAR to antigen density made it possible to maintain anti-tumour activity without recognition of normal tissue. Many other tumours overexpressed TAAs relative to normal tissue as well. Therefore, these methods could be applied to other malignancies.

Another main safety concern was CRS. CRS was characterized by fever, hypoxia and hypotension, and potentially led to organ failure by producing several pro-inflammatory cytokines [51]. Cytokine-blocking drugs like tocilizumab that could block IL-6 signalling pathway and steroids like methylprednisolone were proved to be valid countermeasures [5254]. Moreover, suicide genes such as inducible caspase-9 and herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) were studied [5557]. Also, the use of mRNA-modified T cells in CAR applications might be an ideal approach. A report that was the first preclinical one developed mRNA-modified CART33 cells and showed potent, but transient, in vitro and in vivo activity [58]. This solution would not permanently express CD33-specific CART cells, so it minimized the risk of long-term toxicity.

Challenges and outlook for CAR T-cell therapy

Despite the two tough toxicities, other problems such as minimizing the risk of gene mutation, reducing autoimmune disorders and generating better CAR T products remained challenges for the CAR T-cell therapy. In order to make the CAR T-cell therapies safer, more strategies need to be considered (Figure 2).

As known, modified T cells carried the CAR gene, which is introduced by a retrovirus. However, the target gene is randomly inserted into the genome, so the oncogenes would be activated if the insertion site is located in the proto-oncogene area. It might lead to the high risk of gene mutation and tumorigenicity after T cells reinfused to the body. It was reported that TAL effector nuclease (TALEN) and CRISPR/Cas9 were powerful genome editing tools for safe insertion. Therapeutic transgenes could be inserted to ‘genomic safe harbours’ – chromosomal locations where transgenes would not perturb endogenous gene activity and promote cancer [5961].

Moreover, T cells after gene modification simultaneously carried endogenous TCR and exogenous CAR, both of which might be recombinant resulting in autoimmunity [62]. Fortunately, evidence indicated that pre-treatment with fludarabine and cyclophosphamide, prior to CAR T-cell infusion was helpful to improve the efficiency of cell immunotherapy [63]. However, the study of the immunogenicity would still be in the process and of great significance for patient’s prognosis and the outcome.

In nearly all studies to date, T cells bearing αβ receptors were used [9,64]. However, αβ T cells require specific TAAs and appropriate CMs for activation. Tumour cells would be resistant to αβ T-cell-mediated cytotoxicity when there is loss of TAA expression or absence of CMs [65]. Meanwhile, success with solid tumours was limited [45,66,67]. So, generating better CAR T products seemed to be of great significance. In this regard, biological characteristics and unique functions of γδ T cells that could apply CAR T-cell therapy for solid tumours were highlighted [68]. Rischer et al. [69] suggested that γδ T cells might serve as potent and specific anti-tumour effector cells because they demonstrated that γδ CAR T cells showed cytotoxicity against tumour cell targets. Moreover, Deniger et al. [70] showed that γδ CAR T cells not only killed CD19+ tumour cell lines in vitro, but also inhibited tumour growth in a mouse xenograft model. More recently, another potential advantage of γδ T cells was recommended. Mirzaei et al. [68] suggested that utilizing engineered allogeneic donor-derived γδ T cells that expressed CAR transgene could be theoretically used as an off-the-shelf product because they were not restricted to MHC. All these offered the hypothesis that γδ-derived CAR T-cell product would be a promising therapeutic strategy to improve anti-tumour immune responses.

Also, Curran et al. [71] made T cells genetically modified to constitutively express CD40L with the ability to enhance T-cell proliferation and tumour cell immunogenicity. This approach not only increased CD19-specific CAR/CD40L T cells efficiency but also has profound effects on the tumour micro-environment [71]. Additionally, the incorporation of CMs used in the second- and third-generation CARs could strengthen CAR T-cell activation [3,15,16]. So, we could make efforts to find better combinations of them, which indicated that new generation of CAR T cells might be applied to cancer treatment in the near future.

Conclusions

CAR T-cell therapy is currently perceived as one of the most promising therapeutic approaches for cancer treatment because of significant outcomes in various studies [5,72]. Instead of traditional drug and radiation therapy, CAR T-cell therapy may reduce or even replace some bone marrow transplantations for haematologic malignancies, avoiding high cost of long-term hospitalization and high risk of bone marrow transplantations. However, the application of CAR T-cell therapy still requires efforts and multiple exploratory studies to limit or contain the side effects. Furthermore, cell preparation, functional testing, evaluation of therapeutic efficacy and so on are also required systematic knowledge and norms.

Additionally, some experiments demonstrated that CAR-transduced cytokine-induced killer (CIK) and NK cells were effective [73,74]. To assess the validity, the safety of these approaches appears to be important and necessary. So, we can expect that CIK and NK cells may also be well involved in more clinical CAR therapies in the future.

We believe that with the growing understanding of technology foundations and clinical researches, CAR T-cell therapy will have a promising role in tumour immunotherapy.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81372396].

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • ALL

    acute lymphoblastic leukaemia

  •  
  • AML

    acute myeloblastic leukaemia

  •  
  • BiTE

    bi-specific T-cell engager

  •  
  • CAR

    chimaeric antigen receptor

  •  
  • CEA

    carcinoembryonic antigen

  •  
  • CLL

    chronic lymphocytic leukaemia

  •  
  • CM

    co-stimulatory molecule

  •  
  • CTLA-4

    cytotoxic T lymphocytes-associated antigen 4

  •  
  • EGFR

    epidermal growth factor receptor

  •  
  • HL

    Hodgkin’s lymphoma

  •  
  • HLA

    human leucocyte antigen

  •  
  • HSPC

    haematopoietic stem and progenitor cell

  •  
  • iCAR

    inhibitory CAR

  •  
  • IL

    interleukin

  •  
  • ITAM

    immunoreceptor tyrosine-based activation motif

  •  
  • MCL

    mantle cell lymphoma

  •  
  • MHC

    major histocompatibility complex

  •  
  • MM

    multiple myeloma

  •  
  • NHL

    non-Hodgkin’s lymphoma

  •  
  • NKG2D

    natural killer group 2 member D

  •  
  • NK

    natural killer

  •  
  • PSMA

    prostate specific membrane antigen

  •  
  • ROR1

    receptor tyrosine kinase-like orphan receptor 1

  •  
  • TALEN

    TAL effector nuclease

  •  
  • TCR

    T cell receptor

  •  
  • TCRT

    TCR engineered T cells

  •  
  • TIL

    tumour-infiltrating lymphocyte

  •  
  • TNF

    tumour necrosis factor

  •  
  • TSA

    tumour specific antigens

  •  
  • TSS

    tumour associated antigens

References

References
1
Couzin-Frankel
J.
(
2013
)
Cancer immunotherapy
.
Science
342
,
1432
1433
2
Ruella
M.
and
Kalos
M.
(
2014
)
Adoptive immunotherapy for cancer
.
Immunol. Rev.
257
,
14
38
3
Brentjens
R.J.
and
Curran
K.J.
(
2012
)
Novel cellular therapies for leukemia: CAR-modified T cells targeted to the CD19 antigen
.
Hematology/Am. Soc. of Hematol. Edu. Program
2012
,
143
151
4
Heiblig
M.
,
Elhamri
M.
,
Michallet
M.
and
Thomas
X.
(
2015
)
Adoptive immunotherapy for acute leukemia: new insights in chimeric antigen receptors
.
World J. Stem Cells
7
,
1022
1038
5
Firor
A.E.
,
Jares
A.
and
Ma
Y.
(
2015
)
From humble beginnings to success in the clinic: chimeric antigen receptor-modified T-cells and implications for immunotherapy
.
Exp. Biol. Med.
240
,
1087
1098
6
Suryadevara
C.M.
,
Gedeon
P.C.
,
Sanchez-Perez
L.
,
Verla
T.
,
Alvarez-Breckenridge
C.
,
Choi
B.D.
et al. 
(
2015
)
Are BiTEs the “missing link” in cancer therapy?
Oncoimmunology
4
,
e1008339
7
Stone
J.D.
,
Aggen
D.H.
,
Schietinger
A.
,
Schreiber
H.
and
Kranz
D.M.
(
2012
)
A sensitivity scale for targeting T cells with chimeric antigen receptors (CARs) and bispecific T-cell engagers (BiTEs)
.
Oncoimmunology
1
,
863
873
8
Maude
S.L.
,
Frey
N.
,
Shaw
P.A.
,
Aplenc
R.
,
Barrett
D.M.
,
Bunin
N.J.
et al. 
(
2014
)
Chimeric antigen receptor T cells for sustained remissions in leukemia
.
N. Engl. J. Med.
371
,
1507
1517
9
Kochenderfer
J.N.
,
Dudley
M.E.
,
Kassim
S.H.
,
Somerville
R.P.
,
Carpenter
R.O.
,
Stetler-Stevenson
M.
et al. 
(
2015
)
Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor
.
J. Clin. Oncol.
33
,
540
549
10
Prapa
M.
,
Caldrer
S.
,
Spano
C.
,
Bestagno
M.
,
Golinelli
G.
,
Grisendi
G.
et al. 
(
2015
)
A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing
.
Oncotarget
6
,
24884
24894
11
Pule
M.A.
,
Savoldo
B.
,
Myers
G.D.
,
Rossig
C.
,
Russell
H.V.
,
Dotti
G.
et al. 
(
2008
)
Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma
.
Nat. Med.
14
,
1264
1270
12
Louis
C.U.
,
Savoldo
B.
,
Dotti
G.
,
Pule
M.
,
Yvon
E.
,
Myers
G.D.
et al. 
(
2011
)
Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma
.
Blood
118
,
6050
6056
13
Miao
H.
,
Choi
B.D.
,
Suryadevara
C.M.
,
Sanchez-Perez
L.
,
Yang
S.
,
De Leon
G.
et al. 
(
2014
)
EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma
.
PLoS ONE
9
,
e94281
14
Choi
B.D.
,
Suryadevara
C.M.
,
Gedeon
P.C.
,
Herndon
J.E.
II
,
Sanchez-Perez
L.
,
Bigner
D.D.
et al. 
(
2014
)
Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma
.
J. Clin. Neurosci.
21
,
189
190
15
Kim
M.G.
,
Kim
D.
,
Suh
S.K.
,
Park
Z.
,
Choi
M.J.
and
Oh
Y.K.
(
2016
)
Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics
.
Arch. Pharm. Res.
39
,
437
452
16
Pircher
M.
,
Schirrmann
T.
and
Petrausch
U.
(
2015
)
T cell engineering
.
Prog. Tumor Res.
42
,
110
135
17
Gross
G.
,
Waks
T.
and
Eshhar
Z.
(
1989
)
Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity
.
Proc. Natl. Acad. Sci. U.S.A.
86
,
10024
10028
18
Savoldo
B.
,
Ramos
C.A.
,
Liu
E.
,
Mims
M.P.
,
Keating
M.J.
,
Carrum
G.
et al. 
(
2011
)
CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients
.
J. Clin. Invest.
121
,
1822
1826
19
Kochenderfer
J.N.
,
Dudley
M.E.
,
Feldman
S.A.
,
Wilson
W.H.
,
Spaner
D.E.
,
Maric
I.
et al. 
(
2012
)
B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells
.
Blood
119
,
2709
2720
20
Chmielewski
M.
and
Abken
H.
(
2015
)
TRUCKs: the fourth generation of CARs
.
Expert Opin. Biol. Ther.
15
,
1145
1154
21
Dai
H.
,
Wang
Y.
,
Lu
X.
and
Han
W.
(
2016
)
Chimeric antigen receptors modified T-Cells for cancer therapy
.
J. Natl. Cancer Inst.
108
,
djv439
22
Rafiq S
D.T.
,
Brentjens
R.J.
,
Scheinberg
D.A.
and
Liu
C.
(
2014
)
Engineered T cell receptor-mimic antibody, (TCRm) chimeric antigen receptor (CAR) T cells against the intracellular protein Wilms tumor-1 (WT1) for treatment of hematologic and solid cancers
.
Blood
124
,
2155
23
Chinnasamy
D.
,
Yu
Z.
,
Theoret
M.R.
,
Zhao
Y.
,
Shrimali
R.K.
,
Morgan
R.A.
et al. 
(
2010
)
Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice
.
J. Clin. Invest.
120
,
3953
3968
24
Chinnasamy
D.
,
Tran
E.
,
Yu
Z.
,
Morgan
R.A.
,
Restifo
N.P.
and
Rosenberg
S.A.
(
2013
)
Simultaneous targeting of tumor antigens and the tumor vasculature using T lymphocyte transfer synergize to induce regression of established tumors in mice
.
Cancer Res.
73
,
3371
3380
25
Frigault
M.J.
and
Maus
M.V.
(
2016
)
Chimeric antigen receptor-modified T cells strike back
.
Int. Immunol.
28
,
355
363
26
Watanabe
K.
,
Terakura
S.
,
Martens
A.C.
,
van Meerten
T.
,
Uchiyama
S.
,
Imai
M.
et al. 
(
2015
)
Target antigen density governs the efficacy of anti-CD20-CD28-CD3 zeta chimeric antigen receptor-modified effector CD8+ T cells
.
J. Immunol.
194
,
911
920
27
Till
B.G.
,
Jensen
M.C.
,
Wang
J.
,
Qian
X.
,
Gopal
A.K.
,
Maloney
D.G.
et al. 
(
2012
)
CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results
.
Blood
119
,
3940
3950
28
Savoldo
B.
,
Rooney
C.M.
,
Di Stasi
A.
,
Abken
H.
,
Hombach
A.
,
Foster
A.E.
et al. 
(
2007
)
Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric T-cell receptor for immunotherapy of Hodgkin disease
.
Blood
110
,
2620
2630
29
Di Stasi
A.
,
De Angelis
B.
,
Rooney
C.M.
,
Zhang
L.
,
Mahendravada
A.
,
Foster
A.E.
et al. 
(
2009
)
T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model
.
Blood
113
,
6392
6402
30
Hombach
A.A.
,
Gorgens
A.
,
Chmielewski
M.
,
Murke
F.
,
Kimpel
J.
,
Giebel
B.
et al. 
(
2016
)
Superior therapeutic index in lymphoma therapy: CD30 CD34 hematopoietic stem cells resist a chimeric antigen receptor (CAR) T cell attack
.
Mol. Ther.
24
,
1432
1434
31
Ramos
C.A.
,
Heslop
H.E.
and
Brenner
M.K.
(
2016
)
CAR-T cell therapy for lymphoma
.
Annu. Rev. Med.
67
,
165
183
32
Walter
R.B.
(
2014
)
The role of CD33 as therapeutic target in acute myeloid leukemia
.
Expert Opin. Ther. Targets
18
,
715
718
33
O’Hear
C.
,
Heiber
J.F.
,
Schubert
I.
,
Fey
G.
and
Geiger
T.L.
(
2015
)
Anti-CD33 chimeric antigen receptor targeting of acute myeloid leukemia
.
Haematologica
100
,
336
344
34
Mardiros
A.
,
Forman
S.J.
and
Budde
L.E.
(
2015
)
T cells expressing CD123 chimeric antigen receptors for treatment of acute myeloid leukemia
.
Curr. Opin. Hematol.
22
,
484
488
35
Mardiros
A.
,
Dos Santos
C.
,
McDonald
T.
,
Brown
C.E.
,
Wang
X.
,
Budde
L.E.
et al. 
(
2013
)
T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia
.
Blood
122
,
3138
3148
36
Gill
S.
,
Tasian
S.K.
,
Ruella
M.
,
Shestova
O.
,
Li
Y.
,
Porter
D.L.
et al. 
(
2014
)
Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells
.
Blood
123
,
2343
2354
37
Santoro
S.P.
,
Kim
S.
,
Motz
G.T.
,
Alatzoglou
D.
,
Li
C.
,
Irving
M.
et al. 
(
2015
)
T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression
.
Cancer Immunol. Res.
3
,
68
84
38
Zuccolotto
G.
,
Fracasso
G.
,
Merlo
A.
,
Montagner
I.M.
,
Rondina
M.
,
Bobisse
S.
et al. 
(
2014
)
PSMA-specific CAR-engineered T cells eradicate disseminated prostate cancer in preclinical models
.
PLoS ONE
9
,
e109427
39
Ma
Q.
,
Gomes
E.M.
,
Lo
A.S.
and
Junghans
R.P.
(
2014
)
Advanced generation anti-prostate specific membrane antigen designer T cells for prostate cancer immunotherapy
.
Prostate
74
,
286
296
40
Ruella
M.
and
Levine
B.L.
(
2016
)
Smart CARS: optimized development of a chimeric antigen receptor (CAR) T cell targeting epidermal growth factor receptor variant III (EGFRvIII) for glioblastoma
.
Ann. Transl. Med.
4
,
13
41
Suzuki
M.
and
Cheung
N.K.
(
2015
)
Disialoganglioside GD2 as a therapeutic target for human diseases
.
Expert Opin. Ther. Targets
19
,
349
362
42
Chinnasamy
D.
,
Yu
Z.
,
Kerkar
S.P.
,
Zhang
L.
,
Morgan
R.A.
,
Restifo
N.P.
et al. 
(
2012
)
Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice
.
Clin. Cancer Res.
18
,
1672
1683
43
Jiang
H.
,
Zhang
W.
,
Shang
P.
,
Zhang
H.
,
Fu
W.
,
Ye
F.
et al. 
(
2014
)
Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells
.
Mol. Oncol.
8
,
297
310
44
Blat
D.
,
Zigmond
E.
,
Alteber
Z.
,
Waks
T.
and
Eshhar
Z.
(
2014
)
Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells
.
Mol. Ther.
22
,
1018
1028
45
Morgan
R.A.
,
Yang
J.C.
,
Kitano
M.
,
Dudley
M.E.
,
Laurencot
C.M.
and
Rosenberg
S.A.
(
2010
)
Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2
.
Mol. Ther.
18
,
843
851
46
Caruso
H.G.
,
Hurton
L.V.
,
Najjar
A.
,
Rushworth
D.
,
Ang
S.
,
Olivares
S.
et al. 
(
2015
)
Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity
.
Cancer Res.
75
,
3505
3518
47
Kloss
C.C.
,
Condomines
M.
,
Cartellieri
M.
,
Bachmann
M.
and
Sadelain
M.
(
2013
)
Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells
.
Nat. Biotechnol.
31
,
71
75
48
Fedorov
V.D.
,
Themeli
M.
and
Sadelain
M.
(
2013
)
PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses
.
Sci. Transl. Med.
5
,
215ra172
49
Johnson
L.A.
,
Scholler
J.
,
Ohkuri
T.
,
Kosaka
A.
,
Patel
P.R.
,
McGettigan
S.E.
et al. 
(
2015
)
Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma
.
Sci. Transl. Med.
7
,
275ra22
50
Hudecek
M.
,
Lupo-Stanghellini
M.T.
,
Kosasih
P.L.
,
Sommermeyer
D.
,
Jensen
M.C.
,
Rader
C.
et al. 
(
2013
)
Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells
.
Clin. Cancer Res.
19
,
3153
3164
51
Geldres
C.
,
Savoldo
B.
and
Dotti
G.
(
2016
)
Chimeric antigen receptor-redirected T cells return to the bench
.
Semin. Immunol.
28
,
3
9
52
Maude
S.L.
,
Barrett
D.
,
Teachey
D.T.
and
Grupp
S.A.
(
2014
)
Managing cytokine release syndrome associated with novel T cell-engaging therapies
.
Cancer J.
20
,
119
122
53
Davila
M.L.
,
Riviere
I.
,
Wang
X.
,
Bartido
S.
,
Park
J.
,
Curran
K.
et al. 
(
2014
)
Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia
.
Sci. Transl. Med.
6
,
224ra25
54
Grupp
S.A.
,
Kalos
M.
,
Barrett
D.
,
Aplenc
R.
,
Porter
D.L.
,
Rheingold
S.R.
et al. 
(
2013
)
Chimeric antigen receptor-modified T cells for acute lymphoid leukemia
.
N. Engl. J. Med.
368
,
1509
1518
55
Di Stasi
A.
,
Tey
S.K.
,
Dotti
G.
,
Fujita
Y.
,
Kennedy-Nasser
A.
,
Martinez
C.
et al. 
(
2011
)
Inducible apoptosis as a safety switch for adoptive cell therapy
.
N. Engl. J. Med.
365
,
1673
1683
56
Ciceri
F.
,
Bonini
C.
,
Stanghellini
M.T.
,
Bondanza
A.
,
Traversari
C.
,
Salomoni
M.
et al. 
(
2009
)
Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study
.
Lancet Oncol.
10
,
489
500
57
Wang
W.
and
Wang
Y.
(
2012
)
Equipping CAR-modified T cells with a brake to prevent chronic adverse effects
.
Curr. Gene Ther.
12
,
493
495
58
Kenderian
S.S.
,
Ruella
M.
,
Shestova
O.
,
Klichinsky
M.
,
Aikawa
V.
,
Morrissette
J.J.
et al. 
(
2015
)
CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia
.
Leukemia
29
,
1637
1647
59
Sun
N.
,
Liang
J.
,
Abil
Z.
and
Zhao
H.
(
2012
)
Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease
.
Mol. Biosyst.
8
,
1255
1263
60
Yang
H.
,
Wang
H.
,
Shivalila
C.S.
,
Cheng
A.W.
,
Shi
L.
and
Jaenisch
R.
(
2013
)
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering
.
Cell
154
,
1370
1379
61
Sadelain
M.
,
Papapetrou
E.P.
and
Bushman
F.D.
(
2011
)
Safe harbours for the integration of new DNA in the human genome
.
Nat. Rev. Cancer
12
,
51
58
62
Chhabra
A.
(
2011
)
TCR-engineered, customized, antitumor T cells for cancer immunotherapy: advantages and limitations
.
Scientific World J.
11
,
121
129
63
Ninomiya
S.
,
Narala
N.
,
Huye
L.
,
Yagyu
S.
,
Savoldo
B.
,
Dotti
G.
et al. 
(
2015
)
Tumor indoleamine 2,3-dioxygenase (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs
.
Blood
125
,
3905
3916
64
Jensen
M.C.
,
Popplewell
L.
,
Cooper
L.J.
,
DiGiusto
D.
,
Kalos
M.
,
Ostberg
J.R.
et al. 
(
2010
)
Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans
.
Biol. Blood Marrow Transplant.
16
,
1245
1256
65
Schultze
J.L.
and
Nadler
L.M.
(
1999
)
T cell mediated immunotherapy for B cell lymphoma
.
J. Mol. Med.
77
,
322
331
66
Kershaw
M.H.
,
Westwood
J.A.
,
Parker
L.L.
,
Wang
G.
,
Eshhar
Z.
,
Mavroukakis
S.A.
et al. 
(
2006
)
A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer
.
Clin. Cancer Res.
12
,
6106
6115
67
Lamers
C.H.
,
Sleijfer
S.
,
van Steenbergen
S.
,
van Elzakker
P.
,
van Krimpen
B.
,
Groot
C.
et al. 
(
2013
)
Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity
.
Mol. Ther.
21
,
904
912
68
Mirzaei
H.R.
,
Mirzaei
H.
,
Lee
S.Y.
,
Hadjati
J.
and
Till
B.G.
(
2016
)
Prospects for chimeric antigen receptor (CAR) gammadelta T cells: a potential game changer for adoptive T cell cancer immunotherapy
.
Cancer Lett.
380
,
413
423
69
Rischer
M.
,
Pscherer
S.
,
Duwe
S.
,
Vormoor
J.
,
Jurgens
H.
and
Rossig
C.
(
2004
)
Human gammadelta T cells as mediators of chimaeric-receptor redirected anti-tumour immunity
.
Br. J. Haematol.
126
,
583
592
70
Deniger
D.C.
,
Switzer
K.
,
Mi
T.
,
Maiti
S.
,
Hurton
L.
,
Singh
H.
et al. 
(
2013
)
Bispecific T-cells expressing polyclonal repertoire of endogenous gammadelta T-cell receptors and introduced CD19-specific chimeric antigen receptor
.
Mol. Ther.
21
,
638
647
71
Curran
K.J.
,
Seinstra
B.A.
,
Nikhamin
Y.
,
Yeh
R.
,
Usachenko
Y.
,
van Leeuwen
D.G.
et al. 
(
2015
)
Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression
.
Mol. Ther.
23
,
769
778
72
Teachey
D.T.
,
Lacey
S.F.
,
Shaw
P.A.
,
Melenhorst
J.J.
,
Maude
S.L.
,
Frey
N.
et al. 
(
2016
)
Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia
.
Cancer Discov.
6
,
664
697
73
Chen
X.
,
Han
J.
,
Chu
J.
,
Zhang
L.
,
Zhang
J.
,
Chen
C.
et al. 
(
2016
)
A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases
.
Oncotarget
7
,
27764
27767
74
Tettamanti
S.
,
Marin
V.
,
Pizzitola
I.
,
Magnani
C.F.
,
Giordano Attianese
G.M.
,
Cribioli
E.
et al. 
(
2013
)
Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor
.
Br. J. Haematol.
161
,
389
401
75
Brentjens
R.J.
,
Riviere
I.
,
Park
J.H.
,
Davila
M.L.
,
Wang
X.
,
Stefanski
J.
et al. 
(
2011
)
Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias
.
Blood
118
,
4817
4828
76
Porter
D.L.
,
Levine
B.L.
,
Kalos
M.
,
Bagg
A.
and
June
C.H.
(
2011
)
Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia
.
N. Engl. J. Med.
365
,
725
733
77
Cruz
C.R.
,
Micklethwaite
K.P.
,
Savoldo
B.
,
Ramos
C.A.
,
Lam
S.
,
Ku
S.
et al. 
(
2013
)
Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study
.
Blood
122
,
2965
2973
78
Kochenderfer
J.N.
,
Dudley
M.E.
,
Carpenter
R.O.
,
Kassim
S.H.
,
Rose
J.J.
,
Telford
W.G.
et al. 
(
2013
)
Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation
.
Blood
122
,
4129
4139
79
Lee
D.W.
,
Kochenderfer
J.N.
,
Stetler-Stevenson
M.
,
Cui
Y.K.
,
Delbrook
C.
,
Feldman
S.A.
et al. 
(
2015
)
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial
.
Lancet
385
,
517
528
80
Dai
H.
,
Zhang
W.
,
Li
X.
,
Han
Q.
,
Guo
Y.
,
Zhang
Y.
et al. 
(
2015
)
Tolerance and efficacy of autologous or donor-derived T cells expressing CD19 chimeric antigen receptors in adult B-ALL with extramedullary leukemia
.
Oncoimmunology
4
,
e1027469
81
Porter
D.L.
,
Hwang
W.T.
,
Frey
N.V.
,
Lacey
S.F.
,
Shaw
P.A.
,
Loren
A.W.
et al. 
(
2015
)
Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia
.
Sci. Transl. Med.
7
,
303ra139
82
Garfall
A.L.
,
Maus
M.V.
,
Hwang
W.T.
,
Lacey
S.F.
,
Mahnke
Y.D.
,
Melenhorst
J.J.
et al. 
(
2015
)
Chimeric antigen receptor T cells against CD19 for multiple myeloma
.
N. Engl. J. Med.
373
,
1040
1047
83
Till
B.G.
,
Jensen
M.C.
,
Wang
J.
,
Chen
E.Y.
,
Wood
B.L.
,
Greisman
H.A.
et al. 
(
2008
)
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells
.
Blood
112
,
2261
2271
84
Wang
Y.
,
Zhang
W.Y.
,
Han
Q.W.
,
Liu
Y.
,
Dai
H.R.
,
Guo
Y.L.
et al. 
(
2014
)
Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma treated by CD20-directed chimeric antigen receptor-modified T cells
.
Clin. Immunol.
155
,
160
175
85
Kobayashi
E.
,
Kishi
H.
,
Ozawa
T.
,
Hamana
H.
,
Nakagawa
H.
,
Jin
A.
et al. 
(
2014
)
A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells
.
Biochem. Biophys. Res. Commun.
453
,
798
803
86
Inaguma
Y.
,
Akahori
Y.
,
Murayama
Y.
,
Shiraishi
K.
,
Tsuzuki-Iba
S.
,
Endoh
A.
et al. 
(
2014
)
Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H
.
Gene Ther.
21
,
575
584
87
Schuberth
P.C.
,
Jakka
G.
,
Jensen
S.M.
,
Wadle
A.
,
Gautschi
F.
,
Haley
D.
et al. 
(
2013
)
Effector memory and central memory NY-ESO-1-specific re-directed T cells for treatment of multiple myeloma
.
Gene Ther.
20
,
386
395
88
Mata
M.
,
Vera
J.F.
,
Gerken
C.
,
Rooney
C.M.
,
Miller
T.
,
Pfent
C.
et al. 
(
2014
)
Toward immunotherapy with redirected T cells in a large animal model: ex vivo activation, expansion, and genetic modification of canine T cells
.
J. Immunother.
37
,
407
415
89
Hu
W.X.
,
Chen
H.P.
,
Yu
K.
,
Shen
L.X.
,
Wang
C.Y.
,
Su
S.Z.
et al. 
(
2012
)
Gene therapy of malignant solid tumors by targeting erbB2 receptors and by activating T cells
.
Cancer Biother. Radiopharm.
7
,
711
718
90
Geldres
C.
,
Savoldo
B.
,
Hoyos
V.
,
Caruana
I.
,
Zhang
M.
,
Yvon
E.
et al. 
(
2014
)
T lymphocytes redirected against the chondroitin sulfate proteoglycan-4 control the growth of multiple solid tumors both in vitro and in vivo
.
Clin. Cancer Res.
20
,
962
971
91
Chow
K.K.
,
Naik
S.
,
Kakarla
S.
,
Brawley
V.S.
,
Shaffer
D.R.
,
Yi
Z.
et al. 
(
2013
)
T cells redirected to EphA2 for the immunotherapy of glioblastoma
.
Mol. Ther.
21
,
629
637
92
Lanitis
E.
,
Poussin
M.
,
Hagemann
I.S.
,
Coukos
G.
,
Sandaltzopoulos
R.
,
Scholler
N.
et al. 
(
2012
)
Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor
.
Mol. Ther.
20
,
633
643
93
Huang
G.
,
Yu
L.
,
Cooper
L.J.
,
Hollomon
M.
,
Huls
H.
and
Kleinerman
E.S.
(
2012
)
Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases
.
Cancer Res.
72
,
271
281
94
Krebs
S.
,
Chow
K.K.
,
Yi
Z.
,
Rodriguez-Cruz
T.
,
Hegde
M.
,
Gerken
C.
et al. 
(
2014
)
T cells redirected to interleukin-13Ralpha2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Ralpha1
.
Cytotherapy
16
,
1121
1131
95
Kong
S.
,
Sengupta
S.
,
Tyler
B.
,
Bais
A.J.
,
Ma
Q.
,
Doucette
S.
et al. 
(
2012
)
Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells
.
Clin. Cancer Res.
18
,
5949
5960

Author notes

*

Huan-huan Sha and Dan-dan Wang contributed equally to this work.

This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).