Recent clinical trials utilizing chimeric antigen receptor T cells therapies against solid tumors

Cancer Letters 390 (2017) 188e200

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Recent clinical trials utilizing chimeric antigen receptor T cells therapies against solid tumors Shuanglin Han a, b, Olivier Latchoumanin a, Guang Wu a, Gang Zhou a, Lionel Hebbard a, c, **, Jacob George a, Liang Qiao a, * a b c

Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia Department of Gastroenterology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, Liaoning Province, China Department of Molecular and Cell Biology, James Cook University, Townsville, QLD 4811, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 November 2016 Received in revised form 23 December 2016 Accepted 24 December 2016

Chimeric antigen receptors (CARs) are a series of manufactured receptors that have the capacity of binding to specific antigens expressed on surface of tumor cells. A CAR normally has an extracellular antigen recognition domain derived from single variable chain of a monoclonal antibody, a transmembrane domain and an intracellular T cell activation domain. During the last decade, CAR-T cells were demonstrated to possess great therapeutic effects on hematological malignancies. However, strategies using CAR-T cells to treat solid tumors have been hindered by considerable obstacles including “on tissue off target” effects and cytokine storm syndrome. This review will summarize the current understanding of CAR-T cell therapies and briefly describe the currently enrolled clinical trials in solid tumors. © 2017 Elsevier B.V. All rights reserved.

Keywords: Chimeric antigen receptor Modified T cells Solid tumor Clinical trials Immunotherapy

General introduction on chimeric antigen receptors (CAR) T cells In 1989, Gross, Waks and Eshhar developed geneticallymodified cytotoxic T cells which had the ability to recognize a hapten called 2,4,6-trinitrophenyl. They demonstrated that these genetically-modified cytotoxic T cells could produce interleukin-2 (IL-2) and possessed cytotoxicity [1]. These inspiring results rapidly boosted the development of adoptive cellular immunotherapy for different types of cancers and therefore stimulated the conception of CAR-T cells. CAR-T cells are redirected T cells containing artificially synthetic receptors which are capable of specifically recognizing the antigens expressed on the surface of tumor

Abbreviations: AML, acute myeloid leukemia; APCs, antigen-presenting cells; CAR, chimeric antigen receptor; CTLA-4, cytotoxic-T-lymphocyte antigen 4; CTLs, cytotoxic T lymphocytes; DCs, dendritic cells; ESCs, embryonic stem cells; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; scFv, single chain variable fragment; TCRs, T cell receptors. * Corresponding author. Fax: þ61 2 86273099. ** Corresponding author. Storr Liver Centre, Westmead Institute for Medical Research, University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia; Department of Molecular and Cell Biology, James Cook University, Townsville, QLD 4811, Australia. E-mail address: [email protected] (L. Qiao). http://dx.doi.org/10.1016/j.canlet.2016.12.037 0304-3835/© 2017 Elsevier B.V. All rights reserved.

cells. Recently, anti-CD19-redirected CAR-T cells have shown great effects on patients with B cell malignancies [2,3]. As a novel immunotherapy in cancers, CAR-T cells have several advantages. Firstly, CAR-T cells have the ability to recognize antigens on any human leukocyte antigen background whereas natural T cell receptors (TCRs) expressed on T cell surface need to be matched to the patients' haplotype [4]. Secondly, HLA expression may be downregulated on some tumor cells, and these tumor cells may escape the TCRs-mediated immune response [5]. In contrast, CAR-T cells are still capable of eradicating these escaping tumor cells. Thirdly, CAR-T cells have a much wider range of potential targets on the tumor cells in that not only traditional proteins but also carbohydrates, glycoproteins and glycolipids could form the specific targets for CAR-T cells [6]. Tumor necrosis in response to natural immunity may release inflammatory signals. Subsequently, these signals induce the maturation of the antigen presenting cells known as dendritic cells (DCs) on which B7 superfamily is usually overexpressed. Proteins released by tumor cells are processed by DCs to generate peptides that can bind to major histocompatibility complex-I or major histocompatibility complex-II molecules on DCs, producing peptidebinding MHC (pMHC) which can then bind to specific TCRs, thereby generating the primary signal for T cell activation. At the meantime, ligation of B7 superfamily to CD28 expressed on the

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surface of T cells generates a co-stimulatory signal. Upon receiving these two stimulatory signals, T cells can be fully activated into effector cells and migrate to the tumor sites. CD8þ T cells, also known as cytotoxic T lymphocytes (CTLs), induce apoptosis of tumor cells and CD4þ T cells, known as 'helper' T cells, provide cytokines such as IL-2 to enhance the immune response [7]. According to the natural T cell activation process, a CAR has three major domains: ectodomain, transmembrane domain and endodomain. The ectodomain of a CAR is the antigen recognition domain which is normally composed of single chain variable fragment (scFv), linker and hinge domain. The scFv is derived from the light and heavy chains of immunoglobulins and has the ability to specifically bind to the target antigens and give T cells the primary signal [8]. Between the light chain and heavy chain, there exists a flexible fragment called “linker” which is composed of repeated amino acids imparting the light chain and heavy chain, thereby enhancing the affinity of the CAR to target antigens. The hinge domain is generally derived from CD8 or IgG4, and connects the antigen recognition part with the transmembrane domain. Fig. 1 illustrates the typical structure of a CAR. Inside T cells, both stimulatory signal and co-stimulatory signal are bound to transmembrane domain. Stimulatory signal molecule is typically CD3-z chain which is a part of TCR and is responsible for T cell activation [9]. The most commonly used co-stimulatory signal molecules include CD27, CD28, CD134 (OX40), and CD137 (4-1BB). These molecules provide co-stimulatory signal to T cells activation. Depending on the number of co-stimulatory molecules, CAR-T cells can be divided into three generations. The first-generation CAR-T cells only have stimulatory molecules but no co-stimulatory molecules. The second-generation CAR-T cells have stimulatory molecules and a co-stimulatory molecule. The third-generation CAR-T cells contain stimulatory molecules and more than one costimulatory molecules [10]. Published studies have demonstrated that the second-generation CAR-T cells are obviously better than

189

the first-generation CAR-T cells. However, the targeting efficacy and curative effects of the third-generation CAR-T cells are barely different from that of the second-generation CAR-T cells [11]. Manufacture of CAR-T cells To construct CAR-T cells, primary T cells from specific patients' peripheral blood are collected. In clinical trials, CAR-T cells are routinely generated from CD3þ T cells as the CD3þ population normally represents T cells in the entire blood cells [12]. However, many studies illustrated that the ratio of the specific subpopulation of T cells (e.g., the ratio of CD4þ to CD8þ T cells) might be important in promoting the tumor-eradicating effects and minimizing the toxicity of the activated T cells to normal tissues [13]. Further studies are needed to identify the optimal ratio of CD4þ to CD8þ T cells of the obtained primary T cells so that the tumor-eliminating effects can be maximized while the toxicity to the normal cells can be minimized. The obtained primary T cells need to be activated before being redirected with CAR genes. Primary and co-stimulatory signals are also needed for the activation of the primary T cells. In the activation of primary T cells, special beads coated with anti-CD3/antiCD28 antibodies are broadly used where anti-CD3 antibody provides the primary signal while anti-CD28 provides co-stimulatory signal [14]. Cytokines such as IL-2 can be added into culture system to enhance the activation efficiency [15]. Some studies also used specific APCs such as irradiated K562 tumor cells to help activate T cells [16]. The activated T cells are then genetically modified. In most CART cell therapies, two different delivery systems are used to deliver CAR genes to T cells: viral (including retroviral and lentiviral) and non-viral system. In the first successfully conducted clinical trial [17], a g-retrovirus (named murine leukemia virus) mediated gene delivery was used to treat severe combined immunodeficiency

Fig. 1. Basic structure of a typical CAR.

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(SCID)-X1 in 11 children. By 2013, around 20% of clinical trials used retroviral system to deliver CAR genes into T cells [18]. Lentiviral system is structurally similar to but biologically different from retroviral system in that the lentiviral system is independent of cell division and is more efficient in transfecting non-dividing cells [19]. Comparing with non-viral systems, the viral systems are easy to construct and the viral vectors are capable of inserting exogenous genes into the targeted genome stably and efficiently [20]. In the category of non-viral systems, transposon/transposase systems can be easily used in CAR-T cell manufacture process. Over the recent years, more and more laboratories started using the non-viral delivery systems such as sleeping beauty [21] and piggyBac transposon/transposase system [22] to deliver exogenous genes. These transposon/transposase systems are less costly than the viral systems and are much easier to construct. However, the transfecting efficiency of the non-viral systems is relatively lower than that of the viral systems, making them less ideal in CAR-T cell manufacture. In addition to the above systems, mRNA electroporation has also been used in CAR-T cell generation, however, the instability of mRNA molecule may only permit transient expression of the CAR molecule which severely limits the use of mRNA electroporation during CAR-T cell construction [23]. Following effective transfection, expansion of CAR-T cells is needed before they are injected into patients' blood stream. The method of T cell expansion is similar to the methods of T cell activation. Cytokines or APCs based strategies have been broadly used in T cell expansion, which can result in several hundred-fold [14,24] to several thousand-fold expansion of T cell population [25,26]. It should be noted that the modified T cells should have the ability to proliferate in vivo rather than being fully expanded or differentiated in vitro because the ability of T cells to further proliferate in vivo imparts CAR-T cells the capacity to persist longer in blood stream. It has been reported that patients who received lymphodepleting chemotherapy prior to CAR-T cell therapies experienced more significant tumor regression compared to those without lymphodepletion [27]. Fig. 2 illustrates the typical process of CAR-T cell therapies. Side effects of CAR-T cells Although CAR-T cells have shown great effects in tumor immunotherapy, significant side effects have been observed. The lack of tumor specific antigen may lead to off-target effects in that CAR-T cells could attack normal tissues expressing same antigen as tumor cells, as exemplified by the most successful CAR-T cells against B cell malignancies, where the CAR-T cells were generated against the B cell tumor marker CD19 which is also expressed on

the healthy B cells [28]. Due to B cell aplasia, patients may develop infection for which infusion of exogenous immunoglobulin is usually needed for prophylaxis [29]. The non-specific attack of CART cell therapy is also reflected in a study of metastatic colorectal cancer, where administration of anti-carcinoembryonic antigenredirected CAR-T cells to patients induced severe colitis due to the non-specific targeting of normal colonic tissue [30]. Recent studies have shown that some tumor antigens are also expressed by embryonic stem cells (ESC). As such, CAR-T cells may potentially kill ESCs resulting in impaired self-renewal capacity of patients [31]. Additionally, injecting numerous activated T cells into patients' blood stream can induce a lethal side effect called “cytokine storm syndrome” which is characterized by high fever, hypotension, and even organ failure [32]. In 2014, a clinical trial using anti-CD19redirected CAR-T cells manufactured by Juno Therapies was suspended because of two deaths after transfusing those T cells into patients' blood. Cytokine storm syndrome was highly suspected as the main cause to this tragedy [33]. In some patients, CAR-T cell therapies can also cause tumor lysis syndrome which gives similar symptoms as cytokine storm syndrome. In a clinical trial implemented by the University of Pennsylvania (Identifier: NCT01029366) utilizing anti-CD19-redirected CAR-T cells against chronic lymphoid leukemia, a patient was diagnosed with tumor lysis syndrome on day 22 after CAR-T cells infusion. The main symptoms included fatigue, fever, rigors, diaphoresis, anorexia, nausea, and diarrhea. The patient also developed a significant increase in the blood level of lactate dehydrogenase (1130 U/L) and creatinine (2.60 mg/DL) indicating cellular injury and kidney damage. Fortunately, these abnormalities were normalized with fluid resuscitation and rasburicase treatment [25]. Strategies to promote safety of CAR-T cells Several strategies have been developed to overcome the side effects of CAR-T cell therapies. The first strategy is to insert a suicidal gene into redirected CAR-T cells so that the CAR-T cells may be cleared upon expressing this suicidal gene, and therefore the potential toxicity of CAR-T cells could be avoided (Fig. 3A). Two types of suicidal genes have been used in CAR-T cell therapies: herpes simplex viral thymidine kinase (HSV-TK) gene [34] and the inducible caspase 9 (iCas9) gene [35]. Although suicidal genes could help avoid unwanted cytotoxicity by clearance of CAR-T cells, the tumoricidal efficiency of the CAR-T cells carrying suicidal genes will inevitably be compromised, hence their clinical application is limited. The second strategy is to use the inhibitory CAR (iCAR) (Fig. 3B). This strategy basically relies on taking advantages of the two most

Fig. 2. The typical process of clinical application of CAR-T cells.

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Fig. 3. Strategies to promote safety of CAR-T cells. (A). Inserting the suicidal gene into redirected CAR-T cells would lead to clearance of CAR-T cells via apoptosis after the activation of CART cells. (B1). An iCAR containing a signaling domain of CTLA-4 or PD-1 will cooperate with a CAR targeting the tumor cells expressing the specific tumor antigen. When iCAR-T cells come into contact with the normal tissues, binding of the iCAR with the antigen specifically expressed on the normal tissues will lead to the activation of CTLA-4 or a PD-1 inhibitory signal, preventing the activation of T cells. (B2). Only when the CAR recognizes the antigen expressed on tumor tissue without iCAR binding to normal tissues specific antigen will result in T cell activation. (C1). Recognition of one CAR by one antigen expressed on either tumor tissues or normal tissues only leads to suboptimal T cell activation. (C2). Whereas simultaneous recognition of both antigens on tumor tissues would lead to full activation of T cells, and in normal tissues these two antigens cannot be expressed at the same time.

commonly targeted immune checkpoint molecules: cytotoxic-Tlymphocyte antigen (CTLA-4) and programmed cell death protein 1 (PD-1) [36]. In this scenario, an iCAR encoding signaling domain of CTLA-4 or PD-1 could recognize a normal tissue specific antigen and meanwhile the other CAR could target a tumor specific antigen. Consequently, combination of these two CARs can target the tumor cells expressing the tumor specific antigen, whereas the normal tissues expressing normal tissue specific antigen which could induce the production of signaling domain of CTLA-4 or PD-1 can inactivate the CAR-T cells. In this way, the CAR-T cells can only be activated by tumor cells whereas the cytotoxicity of CAR-T cells to normal tissues can be minimized [37,38]. A third strategy of minimizing the side effects of CAR-T cell therapies has recently been developed. This strategy uses a combinatorial antigen recognition approach (Fig. 3C). In this method, T cells are modified with two CARs in such a way that they can only be minimally activated when binding to one suboptimal tumor-specific antigen. The chimeric co-stimulatory receptor in the other CAR recognizes a second suboptimal tumor-specific antigen. Only when these co-modified T cells recognize tumor cells expressing both antigens can the T cells be fully activated [39]. Moreover, a new technique called Synthetic Notch Receptor has been developed (Fig. 4). In this technique, the extracellular recognition domain of these synthetic receptors was used to detect a large range of cell-surface proteins including tumor-specific antigens. Once the extracellular recognition domain is connected with

specific antigens, the intracellular transcription domain would provide transcriptional signals for specific downstream genes such as a CAR gene, hence allowing the cells expressing the CAR on cell membrane to recognize another targeted antigen. These Synthetic

Fig. 4. Binding of the extracellular domain of the left synthetic receptor to the antigen 1 leads to the activation of the intracellular transcription domain, which releases specific transcriptional signal for the CAR gene leading to the recognition of antigen 2 by the CAR. Hence, full activation of T cells depends on recognition of both antigens.

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Notch receptors enable researchers to detect new input signals in flexible ways and also provide modular output domain, so that the cell sensing and response behaviors can be highly customized, giving a promising strategy to overcome the “on tissue off target” phenomenon of CAR-T cells [40,41]. CAR-T cells in clinical trials for solid tumors Clinical trials have demonstrated excellent outcomes of CAR-T cell therapies in patients with B cell malignancies expressing CD19 [42]. Almost all B cell malignancies highly express CD19, making CD19 an ideal target for CAR-T cell therapy. However, CD19 is also expressed on normal B cells, thus anti-CD19-redirected T cells can potentially target normal B cells. Nevertheless, as the patients lacking B cells can tolerate anti-CD19-redirected T cells for a relatively long time, anti-CD19-redirected CAR-T cells are still believed to be the most successful CAR-T cell therapy for B cell malignancies. So far, CAR-T cell therapies have been attempted in several hematological malignancies including B cell malignancies [43], T cell malignancies [44], acute myeloid leukemia (AML) [45], Hodgkin lymphoma [46], and myeloma [47] with good outcomes. However, multiple hurdles exist for successful CAR-T cell therapies in solid tumors. Firstly, there is a lack of unique tumor antigens that can be used to generate highly specific CAR-T cells. This can lead to severe “on tissue off target” effects, and consequently normal cells or organs may be damaged. Secondly, in solid tumors sites, the activation and effector functions of T cells are impaired by inhibitory molecular pathways including CTLA-4 and PD-1 [48]. Thirdly, inefficient homing of redirected T cells to tumor sites can tremendously lower the therapeutic efficacy. Despite the aforementioned difficulties, the experience and lessons learned from the CAR-T cell therapies in hematological malignancies can facilitate the development of CAR-T therapies against solid tumors. As the redirected T cells lack suitable chemokine receptors to accept the chemokines produced by tumor cells (i.e., lack of chemotaxis), Kershaw et al. transduced the redirected T cells utilizing a retroviral vector encoding C-X-C chemokine receptor 2 and have demonstrated that such genetically modified T cells can migrate to tumor sites secreting specific chemokines [49]. So far, multiple clinical trials on CAR-T cell therapies have been conducted worldwide against several solid tumors such as lung cancer (Identifier: NCT02349724), esophageal cancer (Identifier: NCT02580747), gastric cancer (Identifier: NCT02725125), pancreatic cancer (Identifier: NCT02587689), colon cancer (Identifiers: NCT02617134), breast cancer (Identifiers: NCT02713984), and ovarian cancer (Identifier: NCT02159716), some trials have produced inspiring outcomes. In the following sections, we will summarize some currently registered clinical trials exploring the effect and safety of CAR-T cell therapies against common solid tumors. As no data have been released by these clinical trials, the discussion will mainly focus on the specific tumor antigens and the approaches used to generate the CAR-T cells. The relevant data about these clinical trials are available at clinicaltrials.gov (a service of the U.S. National Institutes of Health).

cancer, biliary malignancy, pancreatic cancer, colon cancer, breast cancer, ovarian cancer and endometrial cancer [55]. In 2009, Carpenito et al. firstly established a mouse xenograft model using the primary cells isolated from the pleural effusion of patients with mesothelioma [56]. The mesothelin-targeted CAR-T cells were then designed and infused into these tumor-bearing immune deficient mice. These CAR-T cells significantly reduced the tumor burden and in some mice the xenograft tumors were completely eradicated [56]. These promising preclinical studies demonstrated the great potential of mesothelin-targeted CAR-T cells in clinical practice. Up to now, several phase 1 clinical trials evaluating the therapeutic effect of mesothelin-targeted CAR-T cells in patients with various cancers have been registered in ClinicalTrials.gov. These trials are currently either in recruiting or ongoing. Among these trials, a phase 1 clinical trial (Identifier: NCT02580747) was initiated to evaluate the therapeutic efficacy of mesothelin-targeted CAR-T cells in patients with relapsed and/or chemotherapy refractory malignancies which are mesothelin positive. Two phase 1 clinical trials evaluating the therapeutic effect, safety and tolerability of mesothelin-targeted CAR-T cells in patients with advanced breast cancer (i.e., metastatic HER2-negative but mesothelin positive breast cancer) (Identifier: NCT02792114) and patients with malignant pleural diseases (including mesothelioma, metastatic breast cancer and lung cancer) (Identifier: NCT02414269) are currently recruiting. In these trials, various approaches for generating and delivering CAR-T cells are used. For example, in the phase 1 trial initiated by Renji Hospital of Shanghai Jiao Tong University (Identifier: NCT02706782), transcatheter arterial infusion (TAI) will be used to deliver the mesothelin-targeted CAR-T cells to the patients with metastatic pancreatic cancer. This approach allows the CAR-T cells to accumulate more in the tumor site than in the normal tissues, thereby the “on-tissue off-tumor” effect is kept to a minimum. In the trial (Identifier: NCT02792114), the mesothelin-targeted CAR-T cells will be directly delivered to patients' pleural cavity. Viral vectors are more commonly used in the generation of mesothelin-targeted CAR-T cells in these clinical trials. For example, in a phase 1 clinical trial looking at the efficacy of the mesothelintargeted CAR-T cells in patients with various metastatic cancers expressing mesothelin (including cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma and lung cancer) (Identifier: NCT01583686), retroviral vector was used in generating CAR-T cells, and in a similar clinical trial (Identifier: NCT02159716), mesothelintargeted CAR-T cells are generated using lentival vectors and the efficacy of the CAR-T cells are to be tested in patients with metastatic pancreatic (ductal) adenocarcinoma, epithelial ovarian cancer and malignant epithelial pleural mesothelioma. As all of the currently registered clinical trials for solid tumors are in the process of recruiting or ongoing and the results from these trials are not yet available, however, we envisage promising outcomes from these trials. Detailed information on the currently registered clinical trials for solid tumors can be found in Table 1 and also on the relevant web sites. Human epidermal growth factor receptor-2 (HER2)

Mesothelin Mesothelin is a glycoprotein located on the cell membrane [50]. The natural function of mesothelin in normal tissues is unclear but recent studies have shown that over-expression of mesothelin on tumor tissues could facilitate cell proliferation, invasion and metastasis, and confer tumor cells with resistance to apoptosis [51e54]. Over-expression of mesothelin has been observed in mesothelioma, thymic cancer, lung cancer, esophageal cancer, gastric

HER2 is a transmembrane protein with tyrosine kinase activity. It is involved in regulating cell survival, proliferation and differentiation through activating PI3K/Akt and Ras/Raf/MEK/MAPK pathways [57]. Previous studies have demonstrated that HER2 is highly expressed in many cancers including cancers of breast [58], lung [59], stomach [60], brain [61] and pancreas [62]. Many preclinical studies have proved the efficacy of HER2-redirected CAR-T cells against HER2 positive cancers. In this aspect, HER2-

Table 1 Summary of the currently registered clinical trials on CAR-T therapies for solid tumours. Targeted markers Targeted cancer types

Phase Age (years) Gender Estimated Status enrollment

Mesothelin

HER2

EGFR

Co-stimulatory signal

Breast cancer; metastatic HER2 breast Malignant pleural disease

1

18

Female 24

Recruiting

1

18

Both

24

Recruiting

Pancreatic cancer

1

18e69

Both

30

Recruiting

Metastatic cancer

1&2

18e70

Both

15

Recruiting

Mesothelin expressing cancers

1

18

Both

21

Unknown

Refractory advanced malignancies HER2þ cancer

1

18e70

Both

20

Recruiting

1&2

18e80

Both

60

Recruiting

Breast cancer

1&2

18e80

Female 60

Recruiting

Glioblastoma

1

18

Both

14

Recruiting

Glioblastoma multiforme

1

All

Both

16

Ongoing

2nd

CD28

HER2þ malignancies

1

3

Both

19

Ongoing

2nd

Retroviral CD28

Metastatic cancer

1&2

18

Both

1

Terminated

Neuroblastoma

1

All

Both

11

Ongoing

3rd

Retroviral CD28&OX40

Neuroblastoma

1

1e18

Both

18

Not yet open 3rd

Retroviral CD28&OX40

Relapsed or refractory neuroblastoma Relapsed or refractory neuroblastoma Relapsed or refractory neuroblastoma GD2þ sarcoma/VEGAS

1&2

1e14

Both

22

Recruiting

1

1

Both

27

Recruiting

2nd

2

1-14

Both

30

Recruiting

4th

1

All

Both

26

Recruiting

GD2þ solid tumors

1

1e35

Both

72

Recruiting

Advanced glioma

1

18e70

Both

10

Recruiting

2nd

4-1BB Retroviral

2nd

Lentiviral 4-1BB

Retroviral

2nd

Retroviral CD28 Retroviral

Retroviral

Notes

TAI drug delivery pathway

Memorial Sloan Kettering Cancer Center, USA Memorial Sloan Kettering Cancer Center, USA Shanghai GeneChem Co., Ltd., China National Cancer Institute (NCI), USA

NCT02792114

Zhujiang Hospital, China

Retroviral

CD28&OX40 Lentiviral

Clinical trial Identifier

Cervical cancer, pancreatic cancer, ovarian cancer and lung cancer University of Pennsylvania, Metastatic pancreatic USA (ductal) adenocarcinoma, epithelial ovarian cancer, malignant epithelial pleural mesothelioma Mesothelin positive tumors Chinese PLA General Hospital, China Glioma, lung cancer, gastric Zhi Yang, Southwest Hospital, China cancer, pancreatic cancer, colorectal cancer, breast cancer and ovarian cancer Fuda Cancer Hospital, Guangzhou, China Baylor College of Medicine, USA HER2 redirected CAR-T cells Baylor College of Medicine, USA that are pre-selected for their ability to recognize cytomegalovirus (CMV) HER2 chimeric receptor and Baylor College of Medicine, TGFbeta dominant negative USA receptor (DNR) expressing EBV specific lymphocytes National Cancer Institute Terminated after the first (NCI) , USA patient’ death due to the treatment iCaspase suicide safety Baylor College of Medicine, switch USA Baylor College of Medicine, Nature killer T cells & USA iCaspase suicide safety switch Sinobioway Cell Therapy Co., Ltd., China Cancer Research UK, UK

Lentiviral

3rd

Sponsor

Patients previously infected with varicella zoster virus (VZV) or previously vaccinated with VZV vaccine Sarcoma, osteosarcoma, neuroblastoma, melanoma

NCT02414269 NCT02706782 NCT01583686

NCT02159716

NCT02580747 NCT02713984

NCT02547961 NCT02442297 NCT01109095

NCT00889954

NCT00924287

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GD2

CAR Vector generation

NCT01822652 NCT02439788

NCT02919046 NCT02761915 NCT02765243

Baylor College of Medicine, NCT01953900 USA

National Cancer Institute (NCI), USA RenJi Hospital, China

NCT02107963 NCT02331693 193

(continued on next page)

Table 1 (continued ) Phase Age (years) Gender Estimated Status enrollment

EGFRvIII

1

18e70

Both

20

Recruiting

1&2

18e65

Both

20

Recruiting

EGFR family

GPC3

CEA

MUC1

Recurrent glioblastoma multiforme EGFR family member positive advanced solid tumor EGFR family member positive advanced solid tumor (lung, liver and stomach) Advanced HCC Advanced HCC

1&2

18e65

Both

20

Recruiting

1 1&2

18e70 18e69

Both Both

20 30

Recruiting Recruiting

GPC3þ HCC

1&2

18e70

Both

60

Recruiting

HCC

1

18

Both

14

Not yet open

Retroviral Lentiviral

Recurrent or refractory lung 1 squamous cell carcinoma CEAþ cancer 1

18e70

Both

20

Recruiting

18e80

Both

75

Recruiting

Liver metastases

1

18

Both

5

Not yet open

Liver metastases

1

18

Both

8

Ongoing

Nasopharyngeal carcinoma 1 and breast cancer Stomach neoplasms 1&2

18e65

Both

30

Recruiting

75

Both

19

Recruiting

Liver Neoplasms

1&2

75

Both

25

Recruiting

MUC1þ solid tumor

1&2

18e80

Both

20

Recruiting

MUC1þ advanced refractory solid tumor

1&2

18e70

Both

20

Recruiting

MUC1þ relapsed or refractory solid tumor

1&2

18

Both

10

Recruiting

1

18

Male

18

Recruiting

1

18

Both

30

Not yet open

Prostate specific Prostate cancer membrane antigen Prostate stem Non-resectable pancreatic cell antigen cancer

2nd

Co-stimulatory signal

4-1BB

Notes

Lentiviral

NCT02844062

PD-1 expressing CAR-T cells Shanghai International Medical Center, China

NCT02862028

TAI drug delivery pathway

RenJi Hospital, China Shanghai GeneChem Co., Ltd., China Fuda Cancer Hospital, Guangzhou, China Baylor College of Medicine, USA Carsgen Therapeutics, Ltd., China Southwest Hospital, China

Roger Williams Medical Center, USA Hepatic artery infusion and Roger Williams Medical Center, USA Yttrium-90 Sir-spheres microspheres Sichuan University, China

Malignant glioma of brain, colorectal carcinoma, gastric carcinoma Hepatocellular carcinoma, non-small cell lung cancer, pancreatic carcinoma, triple-negative invasive breast carcinoma Hepatocellular carcinoma, non-small cell lung cancer, pancreatic carcinoma, triple-negative invasive breast carcinoma, malignant glioma of brain, colorectal carcinoma, gastric carcinoma

Retroviral

Clinical trial Identifier

Beijing Sanbo Brain Hospital, China PD-1 expressing CAR-T cells Ningbo Cancer Hospital, China

Lung cancer, colorectal cancer, gastric cancer, breast cancer, pancreatic cancer Hepatic artery infusion

3rd

Sponsor

NCT02873390

NCT02395250 NCT02715362 NCT02723942 NCT02905188 NCT02876978 NCT02349724

NCT02850536 NCT02416466

NCT02915445

Sinobioway Cell Therapy NCT02725125 Co., Ltd., China Sinobioway Cell Therapy NCT02729493 Co., Ltd., China PersonGen BioTherapeutics NCT02617134 (Suzhou) Co., Ltd., China PersonGen BioTherapeutics NCT02587689 (Suzhou) Co., Ltd., China

PersonGen BioTherapeutics NCT02839954 (Suzhou) Co., Ltd., China

Memorial Sloan Kettering Cancer Center, USA

NCT01140373

T cells genetically modified Bellicum Pharmaceuticals, USA with retrovirus vector containing PSCA-specific CAR and an inducible MyD88/ Cluster Designation (CD)40 (iMC) co-stimulatory domain

NCT02744287

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EpCAM

CAR Vector generation

194

Targeted markers Targeted cancer types

ROR1þ malignancies

1

18

Both

60

T1E28z

Head and neck cancer

1

18

Both

30

Particiption by invitation Recruiting 2nd

VEGFR2

Metastatic cancer

1&2

18e70

Both

24

Completed

IL13Ra2

Recurrent or refractory malignant glioma

1

18e75

Both

75

Recruiting

CD133

Refractory advanced malignancies

1

18e70

Both

20

Recruiting

CD70

CD70þ cancers

1&2

18e70

Both

113

Not yet open

EphA2

EphA2þ malignant glioma

1&2

18e80

Both

60

Recruiting

FAP

FAPþ malignant pleural mesothelioma

1

18e75

Both

6

Recruiting

cMet

Triple negative breast cancer; metastatic breast cancer

1

All

Both

15

Ongoing

CD171

Neuroblastoma; ganglioneuroblastoma

1

18

Both

80

Recruiting

Retroviral

2nd

Lentiviral 4-1BB

Retroviral

Engineered T-cells will be injected directly into the tumour site and patients will not be lymphodepleted Metastatic cancer, metastatic melanoma, renal cancer Malignant glioma, refractory brain neoplasm, recurrent brain neoplasm Liver cancer, pancreatic cancer, brain tumor, breast cancer, ovarian tumor, colorectal cancer, acute myeloid and lymphoid leukemias

FAP-specific redirected T cells given directly in the pleural effusion Autologous cMet redirected T cells administered intratumorally (IT) in patients with breast cancer 2nd & 3rd Lentiviral 2nd generation: T cells are transduced with a lentivirus to express the 4-1BB; 3rd generation: CD171 CAR as well as a truncated EGFR that has no CD28&4-1BB signaling capacity (noted EGFRt)

Fred Hutchinson Cancer Research Center, USA King's College London, UK

NCT02706392 NCT01818323

National Cancer Institute (NCI), USA

NCT01218867

City of Hope Medical Center, USA

NCT02208362

Chinese PLA General Hospital, China

NCT02541370

National Cancer Institute (NCI), USA Fuda Cancer Hospital, Guangzhou, China University of Zurich, Switzland

NCT02830724 NCT02575261 NCT01722149

Abramson Cancer Center of NCT01837602 the University of Pennsylvania, USA Seattle Children's Hospital, USA

NCT02311621

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ROR1

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redirected CAR-T cells were shown to significantly reduce the size of medulloblastoma in the orthotopic and xenogeneic mice model in 2007 [63]. In 2014, animal studies by Mata et al. [64] and Sun et al. [58] proved the efficiency of HER2-redirected CAR-T cells in HER2 positive osteosarcoma and breast cancer, respectively. Consequently, HER2 has become an attractive tumor antigen in the generation of CAR-T cells. Several phase 1 and phase 2 clinical trials have been initiated to investigate the efficacy and safety of HER2-targeted CAR-T cells in patients with various solid cancers including breast cancer (Identifiers: NCT02713984 and NCT02442297) and glioblastoma (Identifiers: NCT02442297 and NCT01109095). Different modifications were applied in the generation of HER2-redirected CAR-T cells in order to increase the targeting specificity and efficacy. For example, in the clinical trial (Identifier: NCT01109095), T cells that can specifically recognize cytomegalovirus (CMV) are selected before being transfected with HER2 CAR. The generated CMV-specific HER2redirected CAR-T cells possess higher targeting activity. In another clinical trial (Identifier: NCT00889954), two genes are inserted into the EBV-specific T cells derived from patients: CAR gene (directed to target HER2) and a dominant negative receptor (DNF) of TGFb signaling (TGFb-resistant). Although safety may be a concern, these T cells will likely exert stronger and more specific antitumor effect in patients with glioblastoma. It should be noted that a phase 1/2 clinical trial on HER2redirected CAR-T cells was terminated following the death of a recruited patient with metastatic cancer that expresses HER2 (Identifier: NCT00924287). In this trial, a 39-year-old female patient developed serious adverse events including decreased platelet count, dyspnea, cardiopulmonary arrest, pleural effusion, anemia, lower gastrointestinal hemorrhage and renal failure. Hence, the safety of HER2-redirected CAR-T cells in the treatment of solid tumors must be carefully addressed.

Disialoganglioside 2 (GD2) GD2 is a glycolipid with a small pentasaccharide head group and is located on the cell membrane. Overexpression of GD2 has been observed in many types of tumor cells [65]. As such, GD2 is regarded as a potential target for immunotherapy in several cancers including neuroblastoma [66], melanoma [67] and osteosarcomas [68]. In a preclinical study, Ming et al. successfully engineered a novel single chain bispecific antibody (hu3F8-scBA) to target GD2, and showed that the engineered hu3F8-scBA has much higher affinity than the other anti-GD2 scFv (5F11-scBA) both in vitro and in vivo. In vivo studies showed that hu3F8-scBA was superior to 5F11-scBA in suppressing tumor growth and prolonging mice survival in the xenograft mouse model of neuroblastoma and melanoma [65]. Clearly, preclinical studies have boosted the development of clinical studies of GD2-redirected CAR-T cells. In two phase 1 clinical trials (Identifiers: NCT02919046 and NCT02761915), normal T cells were used to generate the GD2redirected CAR-T cells, and the efficacy and safety of these CAR-T cells are to be tested in patients with relapsed or refractory neuroblastoma. In the phase 1 clinical trial (Identifier: NCT01822652) for patients with neuroblastoma, a third-generation CAR was used to generate GD2-redirected CAR-T cells. As this CAR contains an iCaspase suicide gene as a safety switch (iC9-GD2-CD28-OX40), and the patients also receive pembrolizumab (a specific inhibitor of PD1), a better efficacy and safety profile is expected. In a similar phase 1 clinical trial in patients with neuroblastoma (Identifier: NCT02439788), natural killer T cells rather than the normal T cells are transfected with the GD2-redirected CAR (iC9-GD2-CD28OX40). This modification allows investigators to compare the

efficacy of the natural killer T cells and normal T cells in CAR-T cell therapies. Recently, a fourth-generation CAR redirected to GD2 (4SCARGD2) was generated, and its efficacy and safety in patients with refractory and/or recurrent neuroblastoma are to be tested in a phase 2 clinical trial (Identifier: NCT02765243). This is the only clinical trial testing the efficacy and safety of a fourth-generation CAR-T cells therapy. In a recently registered phase 1 clinical trial (Identifier: NCT01953900), T cells were obtained from the sarcoma patents who were either previously infected with varicella zoster virus (VZV) or were previously vaccinated against VZV. This trial aims to test the therapeutic efficacy and safety of GD2-redirected CAR-T cells (CARiC9-GD2-CAR-VZV-CTLs) in sarcoma patients, and the possible impact of VZV vaccination on the in vivo expansion and persistence of the generated CAR-T cells. Constant technological improvement is needed to enhance the efficacy and safety of CAR-T cells therapies. A third-generation CART cell therapy was recently registered in a phase 1 clinical trial (Identifier: NCT02107963). In this study, GD2-redirected CAR contains a caspase dimerization domain (ICD9) as a suicide switch, and the generated CAR-T cells are believed to be safer. This trial will test the efficacy and safety of GD2-redirected CAR-T cells in patients with sarcoma, osteosarcoma, neuroblastoma and melanoma in whom GD2 is positive. Epidermal growth factor receptor (EGFR) EGFR is a cell membrane surface receptor that belongs to ErbB family of receptors. Binding of EGFR by its ligands induces phosphorylation of receptor tyrosine kinase and activation of downstream signal transduction pathways, consequently regulating the proliferation and differentiation of the target cells. Many studies have confirmed the overexpression of EGFR in numerous types of tumor cells, and the expression level of EGFR is adversely associated with patients' prognosis and survival. In addition, EGFR is mechanistically linked to the resistance of cancer cells to chemotherapy and radiation treatment [69]. Preclinical studies testing the effect of EGFR-redirected CAR-T cells in patients with glioblastoma have been reported. For example, intracerebral injection of thirdgeneration CAR-T cells targeting epidermal growth factor receptor variant III (EGFRvIII, a mutated form of EGFR) in patients with glioma led to reduced tumor growth [70]. In an xenograft mouse model of glioblastoma, intravenous instead of intracerebral injection of the same third-generation EGFRvIII-redirected CAR-T cells also resulted in accumulation of the therapeutic CAR-T cells in the intracerebral tumor site, and marked reduction in tumor growth and improvement of animal survival were reported [71]. These promising results triggered further clinical trials. A phase 1 clinical trial testing the efficacy and safety of EGFRredirected CAR-T cells in patients with advanced glioma has been registered (Identifier: NCT02331693). In another recently registered phase 1 clinical trial (Identifier: NCT02844062), EGFRvIII was used as the specific tumor antigen in the generation of CAR. The efficacy and safety of the EGFRvIII-redirected CAR-T cells are to be tested in patients with recurrent glioblastoma. The therapeutic effects of EGFR-redirected CAR-T cells are also to be tested in other malignant tumors in clinical trials. In a phase 1/2 clinical trial (Identifier: NCT02873390), the EGFR-redirected T cells were designed to contain PD-1 gene in order to enhance the safety of the CAR-T cells. This trial aims to test the efficacy and safety of the EGFR-redirected CAR-T cells in patients with advanced malignancies in whom the tumor tissues must be positive for at least one protein from EGFR family (including EGFR, HER2, HER4), EGFRvIII and IGF1R. The same EGFR-redirected CAR-T cells are also to be

S. Han et al. / Cancer Letters 390 (2017) 188e200

tested in a phase 1/2 clinical trial (Identifier: NCT02862028) in patients with cancers of lung, liver and stomach in whom EGFR is positive. Glypican-3 (GPC-3) Glypican-3 (GPC-3) is heparan sulfate proteoglycan that belongs to the glypican family which consists of six subtypes (GP 1-6). GPC3 is connected to extracellular surface of cell membrane by a glycosyl-phosphatidylinositol anchor [72], and is involved in the regulation of multiple cell behaviors such as migration, invasion and apoptosis possibly via interacting with Wnt and Hedgehog pathways [73]. Studies have shown that GPC-3 is positive in all hepatocellular carcinoma (HCC) tissues but is negative in the dysplastic nodules [74]. Importantly, GPC-3 is not expressed in normal adult liver [75]. These features make GPC-3 a potential specific biomarker for HCC in terms of early diagnosis and therapeutic targeting. In a preclinical study in mice carrying xenograft human HCC, the third-generation GPC-3-rdirected CAR-T cells could eliminate the HCC xenografts in which GPC-3 expression was high and suppress the growth of HCC xenografts in which GPC-3 expression was low. Additionally, with these CAR-T cells could significantly extend the animal survival [76]. Four phase 1/2 clinical trials (Identifiers: NCT02395250, NCT02715362, NCT02723942, and NCT02905188) have been registered to test the therapeutic efficacy and safety of the GPC-3redirected CAR-T cells in HCC patients. As of the time this manuscript is prepared, the trial NCT02905188 is not yet open but the other three trials are recruiting. As the efficacy of the GPC-3-redirected CAR-T cells have been tested in human xenograft liver tumors in mice [76], and overexpression of GPC-3 was also found lung squamous cell carcinoma [77], a phase 1 clinical trial has been registered (Identifier: NCT02876978) in which GPC-3-redirected CAR-T cells are to be tested for their appropriate dosing, efficacy and safety in patients with lung squamous cell carcinoma.

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the therapeutic efficiency can be enhanced and toxicity would be minimized. In order to improve the therapeutic efficacy of CEA-redirected CAR-T cells in patients with metastatic liver cancer, another clinical trial has been registered (Identifier: NCT02416466) in which the CEA-redirected CAR-T cells are to be given to patients together with the arterially delivered yttrium-90 SIR-Spheres, an approved internal radiation therapy for metastatic liver cancer. Epithelial cell adhesion molecule (EpCAM) EpCAM is a transmembrane glycoprotein and plays an important role in mediating Ca2þ independent adhesion of epithelial cells. EpCAM has been shown to induce proliferation and enhance cellular metabolism via up-regulating c-myc, cyclin A and cyclin E [81]. EpCAM is believed to be a reliable marker for cancer stem cells in many cancers. Over the recent years, EpCAM has been shown to be a potential therapeutic target for many cancers of epithelial origin. Of more relevance to this review, EpCAM has been used as a tumor antigen in CAR-T cells therapies. The therapeutic effect of EpCAM-redirected CAR-T cells has been tested in animal models of prostate cancer [82]. The study revealed that the EpCAM-redirected CAR-T cells could inhibit the growth of tumors harboring high level of EpCAM. The efficacy of EpCAM-redirected CAR-T cells was also tested in a metastasis model of prostate cancer in which the growth of the tumors expressing low level of EpCAM was also suppressed and the animal survival was extended by the EpCAM-redirected CAR-T cells, possibly due to the selective targeting of the subpopulation of metastatic cancer cells [82]. Several phase 1 clinical trials have been registered to test the efficacy and safety of EpCAM-redirected CAR-T cells in patients with EpCAM positive nasopharyngeal carcinoma and breast cancer (Identifier: NCT02915445), gastric cancer (Identifier: NCT02725125) and liver cancer (Identifier: NCT02729493). These trials are currently in recruiting. Mucin1 (MUC1)

Carcinoembryonic antigen (CEA) CEA is a glycoprotein serving as the ligand of L-selectin and Eselectin in colon cancer. Expression of CEA is closely related to the metastasis of colorectal cancer [78]. Physiologically, CEA is produced by gastrointestinal tissues during the development of fetus and its level would greatly decline after birth. Hence, CEA is barely detectable in adult serum. In patients with colorectal carcinoma, the serum level of CEA may raise sharply. Based on these properties, CEA has been used as a potential diagnostic and prognostic marker for patients with colorectal cancer [79]. In addition, CEA may be a potential therapeutic target. A recent preclinical study suggested that CEA-redirected CAR-T cells may hold potential for patients with CEA positive solid tumors [80]. However, as CEA is also expressed in healthy tissues, the safe dosage of the CEA-redirected CAR-T cells in cancer patients must be carefully determined [80]. A phase 1 clinical trial has been registered (Identifier: NCT02349724) in which the efficacy and safety of the CEAredirected CAR-T cells will be evaluated in patients with several CEA positive cancers including lung cancer, colorectal cancer, gastric cancer, breast cancer and pancreatic cancer. In another registered but not yet open phase 1 clinical trial (Identifier: NCT02850536), CEA-redirected CAR-T cells are to be tested in patients with CEA positive metastatic liver cancer. In this trial, the CEA-redirected CAR-T cells are given to patients by percutaneous hepatic artery infusion using the “Surefire Infusion System (SIS)”. With this highly accurate delivery system, it is expected that the CAR-T cells would highly accumulate into tumor sites, and therefore

MUC1 is a glycosylated phosphoprotein anchored to the surface of epithelial cells by a transmembrane domain. MUC1 protects the epithelium from non-MUC1 binding bacteria via steric hindrance effect that restrains the bacteria from attaching onto the cell surface. Additionally, MUC1 could directly bind MUC1-binding bacteria hence preventing the bacteria from their detrimental effect on epithelium [83]. MUC1 is expressed in almost all normal epithelial cells, but it is overexpressed and aberrantly glycosylated in most epithelial cancers [84]. Many preclinical studies were conducted to test if MUC1 could be a potential therapeutic target. In a recently published preclinical study in T cell leukemia and pancreatic cancer [85], an abnormal glycoform of MUC1 named Tn-MUC1 was used as the target molecule. Tn-MUC1-redirected CAR-T cells showed specific cytotoxicity to cancer cells expressing Tn-MUC1 and eventually inhibited the growth of the xenograft tumors in mice [85]. These promising outcomes make MUC1 and its variants potentially useful targets for cancer therapy. Currently, three phase 1/2 clinical trials have been registered to investigate the efficacy and safety profiles of MUC1-redirected CART cells in patients with malignant glioma, colorectal carcinoma, and gastric carcinoma (Identifiers: NCT02617134), patients with refractory solid tumor including HCC, non-small cell lung cancer, pancreatic carcinoma, and triple-negative invasive breast carcinoma (Identifier: NCT02587689), and patients with all of these solid tumors but the NK cells instead of T cells were used to generate the MUC1-redirected CAR-T cells (Identifier: NCT02839954). The outcomes of these studies are pending.

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Other targeted markers

References

Apart from the aforementioned markers, a number of other cell surface markers have been attempted as potential target in CAR-T cells therapies, as detailed in Table 1. So far, 49 clinical trials on CAR-T cell therapies for solid tumors have been registered but only one trial was completed in 2016. In this completed trial (Identifier: NCT01218867) implemented by National Cancer Institute (NCI), vascular endothelial growth factor receptor 2 (VEGFR2) was used as the tumor marker in the development of CAR-T cells. These VEGFR2 redirected CAR-T cells were used to target metastatic melanoma and renal cancer. Although initial outcomes have been released, statistic data on the therapeutic efficacy, adverse effects, and patient survival are not yet available.

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Conclusion and prospect This review summarizes 49 clinical trials. Most of these trials were or are being carried out in USA (23/49) and China (23/49), two in UK and one in Switzerland. Majority of the trials (41/49, 83.7%) are either ongoing or recruiting patients. Five registered clinical trials are not yet open, one trial remains unknown, one was terminated because of the death of a participant. Only one trial has completed so far. Therefore, conclusions for these trials can not be reached at this stage. Worldwide, considerable progress has been made in the evaluation of the therapeutic efficacy and safety of CAR-T cells in hematological malignancies especially in B cell malignancies. However, little progress has been made in the exploration of CAR-T cell therapies in solid tumors. Lack of specific targetable tumor antigen appears to be a key factor responsible for the poor specificity and poor efficacy of CAR-T cell therapies in solid tumors. Lack of specific tumor markers likely contribute to the “on tissue off target” effect, and life-threatening adverse effects such as cytokine storm syndrome and tumor lysis syndrome. Technic improvement and modification of the existing various CAR-T cells have already achieved promising results in the treatment of solid tumors. These technic improvement or modifications include inserting suicidal genes, combining immune checkpoints molecules, using combinatorial antigens, and creating synthetic Notch receptor system as discussed above. Persistent search for more specific tumor surface antigens and extensive in vitro and animal studies should be conducted to improve the specificity, efficacy and safety of CAR-T cells in cancer therapy. Lastly, combinatorial effect of CAR-T cells with other existing therapeutic approaches should be explored. With extensive in vitro and in vivo studies, and more properly designed clinical trials, we envisage that in the future, CAR-T cell therapies would become a powerful and potentially curative approach for solid tumors. Acknowledgments Dr. L Qiao's work was supported by the Robert W. Storr Bequest to the Sydney Medical Foundation, University of Sydney, a National Health and Medical Research Council of Australia (NHMRC) Project Grant (ID: 1047417) and a Cancer Council NSW grant (ID: APP1070076). LH is funded by Project Grants from the Cancer Council New South Wales APP1069733 Queensland APP1123436 and a James Cook University Development Grant 94066. Conflict of interest The authors declare no conflict of interest.

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