Chimeric antigen receptor T cells: a novel therapy for solid tumors

Glioblastoma (GBM) remains one of the deadliest primary brain tumors in adults, and standard treatments for GBM do not significantly increase the survival time. EGFRvIII is expressed in GBM cell surface; therefore, CAR-T cell targeting EGFRvIII is a novel strategy worth studying [47]. Morgan et al. conducted a series of experiments to construct competent CARs and evaluated the ability of CAR-engineered T cells recognizing EGFRvIII. Considering that the established cell lines may not keep the molecular characteristics of primary human cancers, Morgan group selected the glioblastoma stem cells (GSCs) expressing EGFRvIII as target cell lines. CAR scFv derived from human mAb 139 recognized GSCs expressing mutant EGFRvIII, but not human normal tissues. T cell signaling transduction domain CD28-41BB-CD3ζ (named 139-28BBZ) made CAR-T keep better survival comparing with the original CAR vector that used CD28-CD3ζ (named 139-28Z) [48‐50], but the biological activity and cytotoxicity were at the equal level. The engineered T cells expressing CAR can specifically recognize EGFRvIII+ cell lines, while no reactivity to co-cultured normal tissue cells. At present, a phase I clinical trial (NCT01454596) using anti-EGFRvIII CAR-T cells is recruiting patients with recurrent glioblastoma [51]. Study by Marcela et al. also evaluated the characteristics of anti-EGFRvIII CAR-T cells and verified its antitumor activity against glioblastoma cells in vitro and vivo [52]. The humanized anti-EGFRvIII CAR-T cells produced IFN-γ, IL-2, TNF-α, and only lysed EGFRvIII-expressing target cells. In order to confirm the antitumor activity in vivo, U87-EGFRvIII tumors were implanted subcutaneously and intracranially into NSG mice, respectively. The results indicated that CAR-T-EGFRvIII cells controlled tumor growth and increased median survival time. This group also used mice grafted with normal human skin to test the potential toxicities of anti-EGFRvIII CAR-T cells, and the results of skin graft assay demonstrated that no significant lymphocytic infiltrate by immunohistochemistry. On this basis, Marcela group started a phase 1 clinical trial (NCT02209376) of EGFRvIII-specific CAR-T cells in patients with either residual or recurrent glioblastoma [53]. D-270MG is a tumor cell line that naturally expresses EGFRvIII [54]. Sampson et al. established the D-270MG^FLuc/GFP subline that co-expressed firefly luciferase (FLuc) and GFP as the target of EGFR-specific CAR-T cells. The study results demonstrated that anti-EGFRvIII CAR-T cells effectively transpassed the blood-brain barrier (BBB) to arrive at invasive GBM tumors and mediated tumor regression and prolonged survival in NSG mice [55]. Zuo et al. used the EGFR-positive (EGFR+) cells including A549, NCI-H1299, NCI-H460, SGC7901, HT29, and EGFR-knockdown (EGFR−) cells including A549-EGFR−, SGC7901-EGFR−, and HT-29-EGFR− to investigate the antitumor activity of EGFR-specific CAR-CIK cells. The study reported that EGFR-specific CAR observably potentiated cytotoxicity and induced secretion of IFN-γ and IL-2 in EGFR-positive cell lines and xenograft tumor models, but not in EGFR negative ones [56]. In summary, the preclinical studies of EGFR-specific CAR-T cells exhibited potent antitumor effect in vitro and in vivo.

Multicenter clinical trials using CAR-T cells targeting EGFR or EGFRvIII are underway. We summarized these clinical trials in Table 2. A phase I trial by Han et al. studied the EGFR-targeted CAR-T cells in 11 patients with EGFR-expressing advanced relapsed/refractory NSCLC (NCT01869166). In this study, the six female and five male patients were divided into three cohorts: in cohort 1, EGFR-CAR T cells infused into four patients directly without any conditioning regimens; in cohort 2, two patients were conditioned with cyclophosphamide, followed by CAR-T EGFR therapy; and in cohort 3, two patients were conditioned by cyclophosphamide, pemetrexed, and cisplatin and three were conditioned by cyclophosphamide, docetaxel, and cisplatin, respectively. All patients received EGFR-targeted CAR-T cell infusions at dose ranged from 0.45 to 1.09 × 10⁷ cells/kg. Of 11 patients, there were two persons acquired PR and five kept stable disease (SD). The anti-EGFR CAR-T cells secreted cytokines including IL-2, IL-4, IL-6, IFN-γ, TNF-α, GM-CSF, and granzyme B in co-culture with EGFR-positive tumor cells. However, after the infusion of EGFR-specific CAR-T cells, the serum levels of cytokines observed at different time point were less obvious compared with the experiment in vitro. Investigators monitored the copy numbers of CAR-EGFR transgene in peripheral blood (seven patients) and tumor tissues (four patients) by quantitative real-time PCR. In peripheral blood, the copy numbers of CAR-EGFR transgene hold high level for more than 4 weeks. CAR-EGFR transgene specifically accumulated in tumor tissues. The only tolerable and controllable toxicities reported in the study were skin toxicity, nausea, vomiting, dyspnea, and hypotension, and there was no cytokine storm observed. Therefore, the CAR-T-EGFR cells were found to be feasible and safe in patients of relapsed/refractory NSCLC [57].

In GBMs, CD133-positive stem cells keep higher expression of HER2 than CD133-negative counterparts. A study result indicated that HER2-specific CAR-T cells targeted and killed autologous HER2-positive GBMs in vitro and facilitated regression of GBMs in an orthotopic xenograft model [71]. Sun et al. constructed a humanized HER2 CAR-T cell containing chA21scFv and examined its antitumor activity. The results indicated that chA21-28z HER2-specific CAR-T cells recognized and killed HER2+ breast and ovarian cancer cells in vitro. Simultaneously, abundant IFN-γ and IL-2 secretion were also detected. In xenograft model, the HER2-specific CAR-T cells also significantly restricted tumor growth [72]. Another study demonstrated that oligoclonal camelid single-domain antibodies (VHHs) could target a range of different epitopes on HER2 antigen. Based on the potent targeting ability of oligoclonal VHHs, the oligoclonal VHH_HER2-CAR-engineered Jurkat T cells exhibited higher expansion, cytokine secretion, and cytotoxicity when exposed to HER2-expressing cells [73]. To reduce antigen escape, Hegdeet et al. created a bispecific CAR molecule co-targeting the two glioma-associated antigens, HER2 and IL-13Rα2, and expanded the CAR-T cells expressing tandem CARs (TanCAR). Encouragingly, the TanCAR effectively redirected T cells to the two antigens and enhanced the function of CAR-T cells and the secretion of cytokines in vitro and in vivo. Therefore, the TanCAR-T cell agents were considered as a potential therapeutic method to control tumor growth as this study reported [74, 75]. Recently, a group combined bispecific antibody αHER2/CD3 and CAR-T therapy. Their data indicated that αHER2/CD3 RNA-engineered T cells exhibited antitumor activity in HER2⁺ N87 tumor cells and in N87 tumor-bearing mice. Moreover, bystander T cells also showed the similar effects. This new strategy may be a potential therapeutic approach for HER2⁺ malignancies [76]. To promote the transduction efficiency, EBV-CTLs were modified to express HER2-CAR via the nonviral piggyBac (PB) transposon which had high gene-transfer efficiency and large coding capacity. PB-modified HER2-CTLs could specifically target and kill HER2-positive tumor cells in vivo and suppress tumor growth in xenogeneic murine models [77]. Although 60% human osteosarcoma expressed HER2 [62, 78], a low level of HER2 renders monoclonal antibodies to HER2 ineffective. Hence, a group used genetic-modified T cell targeting HER2 to determine the antitumor activity in osteosarcoma. The HER2-specific CAR-T cells proliferated, produced cytokines, and killed tumor cells after exposure to HER2-positive osteosarcoma cell lines in vitro. Moreover, they created two mouse models: one is locoregional disease in a severe combined immune deficiency (SCID) mouse model and the other is lung metastases model. Adoptive transfer of HER2-specific CAR-T cells caused osteosarcoma regression at the different sites [79]. Similarly, HER2-specific CAR-T cells had the capacity of recognizing and killing HER2-positive medulloblastoma cells in vitro and induced regression of tumors in an orthotopic xenogeneic SCID model [64]. These preclinical studies have achieved encouraging results, promoting HER2-specific CAR-T clinical trials to test the feasibility and safety.

At present, Southwest Hospital in China, Chinese PLA General Hospital, Fuda Cancer Hospital Guangzhou, and Baylor College of Medicine are carrying out clinical trials of HER2-specific CAR-T cells. We summarized these clinical trials in Table 2. Phase I/II clinical study (NCT00924287) sponsored by National Cancer Institute (NCI) has completed. This trial was designed to evaluate the safety and efficacy of HER2-specific CAR-T cells in patients with relapsed/refractory HER2-positive sarcoma. Nineteen patients received escalating doses (range 1 × 10⁴/m² to 1 × 10⁸/m²) of HER2-specific CAR-T cells including eight dose levels. The study reported that among the detected serum cytokines, only the concentration of IL-8 had significantly increased within 1 week after infusion and persisted for up to 4 weeks. Although HER2-specific CAR-T cells had no expansion after infusion in the peripheral blood, these cells could traffic to tumor sites and maintain at low levels for more than 6 weeks. T cell persistence and copy number were correlated with the infused T cell dose. The clinical benefit of HER2-specific CAR-T cell was not encouraging, only four of nineteen patients acquired stable disease (SD). In the process of HER2-specific CAR-T cell infusion, dose-limiting toxicity was not observed apart from a patient with the highest dose levels within 12 h post-infusion [80].

June et al. demonstrated that the mesothelin-specific T cells exhibit antitumor effects on large pre-established mesothelioma xenografts in NOD/scid/IL2rγ^−/− mice. Their data suggested that the combination of CD137 and CD28 improved multifunctional cytokine secretion and enhanced the function of CAR T cells in tumor-bearing mice [103]. In the tumor microenvironment, some inhibitors hampered the function of CAR T cells. For example, diacylglycerol kinase (dgk), as a negative regulator of TCR signaling, is expressed in T cells. Its isoform includes dgkα and dgkζ. Previous studies found that deletion of either dgk isoform induced the activation of DAG-mediated Ras/ERK pathway and proliferation of T cells [104‐106]. Based on this, Koretzky et al. demonstrated that deletion of dgks greatly enhanced activity against tumor and improved persistence of CAR-engineered T cells targeting mesothelin in vitro and in implanted tumors. Beyond that, pharmacologic inhibition of dgks also facilitated function of mesothelin-specific CAR-T cells. Moreover, dgk-deficient T cells showed decreased sensitivity to TGFβ and increased FasL and TRAIL expression. Such a combined therapeutic approach might be translated clinically as the study reported [107]. Moon et al. found that a single intravenous injection of human mesoCAR-T cells into immunodeficient mice significantly restrained the tumor growth but did not cure tumor. They considered that upregulation of inhibitory receptors was the main cause of mesoCAR-T cell hypofunction [108]. As an inhibitor within the tumor microenvironment, upregulation of PD-1 limited T cell function [109]. Cherkassky et al. found that PD-1 antibody could reverse PD-1-mediated CAR-T cell exhaustion and mesoCAR-T cells also showed delayed exhaustion upon repeated antigen stimulation. Hence, combination of costimulation and cell-intrinsic PD-1 checkpoint blockade could overcome inhibitory effect on CAR-T cells in MSLN-expressing tumor microenvironment [110]. CAR-T therapy achieved good results in preclinical studies. But the effect was not satisfied in the clinical trials mainly due to its adverse effects. For example, scFv was generally derived from murine monoclonal antibodies; the induction of human anti-mouse antibody (HAMA) might shorten T cell survival time [111]. A study demonstrated that fully human mesothelin-specific CAR-T cells showed potent cytolytic activity toward mesothelin-positive tumor cells and controlled large, well-established ovarian cancer growth in a xenogeneic mouse model. Besides, mesothelin-specific CAR-T cells induced bystander killing of mesothelin-negative tumor cells [112]. On-target/off-tumor toxicity could cause life-threatening adverse effects in the application of CAR-T cells, because the target antigen also expressed on normal cell surface at low levels. Both a-folate receptor (FRa) (90%) and mesothelin (70%) were overexpressed in ovarian cancers [113, 114], and their expression pattern on normal tissues is mainly non-overlapping. Based on the foundation of above studies, Daniel et al. generated trans-signaling CAR T cells engineered to co-express anti-mesoscFv-CD3 and anti- FRascFv-CD28CARs, aiming to diminish the potential toxicity of CAR-T cells to normal tissue cells expressing low levels of TAAs. The result indicated that trans-signaling CAR-T cells exhibited higher antitumor potential in vitro and in vivo. Moreover, trans-signaling CAR-T cells were resistant to antigen-induced cell death (AICD) [115]. The successes achieved by CAR-T cells in hematological malignancies were unable to be accomplished in solid tumor, partly owing to the low efficacy of CAR-T cells homing to tumor sites. Stimulating more chemokine receptors expressed on CAR-T cells or direct regional injection may be valid. Chemokine CCL2 is highly expressed by MPM tumors, but the expression level of CCL2 receptor CCR2 on resting and activated T cells is low. Therefore, Moon et al. transduced the chemokine receptor CCR2b into mesoCAR-T cells to potentiate trafficking of CAR-T cells into tumors. Their study demonstrated that the functional CCR2b in the mesoCAR-T cells significantly increased the number of intratumoral T cells and improved antitumor efficacy in vitro and in vivo [116]. Adusumilli et al. found that compared with intravenous injection, intrapleural administration of anti-mesothelin CAR-T cells exhibited greater antitumor potency and strongly promoted the expansion, differentiation, and persistence of T cells [117].

Many clinical trials about mesothelin-specific CAR-T cells are ongoing. We summarized these clinical trials in Table 2. Marcela et al. started a clinical study in four patients infused with autologous T cells transducted with mRNA to express CAR derived from a murine antibody to human mesothelin. These results demonstrated that when patients received intermittent infusion of meso-RNA CAR-T cells, the serum IgE levels detected via ELISA assay were elevated which caused anaphylaxis. Therefore, they suggested that a single infusion of stably transducted, long-lived CAR-T cells or constructing CAR based on the humanized antibodies may be safer and more effect [52]. The phase I clinical trial (NCT01355965) conducted by Beatty et al. was designed to improve the feasibility and safety of mRNA-transduced CAR-T cells targeting mesothelin (mesoCAR-T cells) in patients with advanced MPM. They presented two case reports indicating that mRNA CAR-T cells showed potent antitumor activity without evident on-target/off-tumor toxicity against normal tissues, infiltrated solid tumor tissues, and induced humoral epitope spreading after infusion [118].