Chimeric antigen receptor T-cell therapy in glioblastoma: charging the T cells to fight

Glioblastoma (GBM), WHO grade IV glioma, is the most devastating brain tumor in adults [1]. The intrinsic capability of single tumor cells to invade normal brain tissue impedes surgical eradication, predictably resulting in early local recurrence and death. Current treatment options for GBM include maximal surgical removal, radiotherapy and chemotherapy. However, the overall survival rate has not changed significantly over the past decade.

Adoptive cell transfer (ACT) using autologous lymphocytes for treating cancer started in 1988 with metastatic melanoma patients [2]. While early trials reported an overall response in approximately 34% of patients [3], subsequent studies demonstrated that lymphodepletion prior to ACT may significantly improve the response rates to about 50% of patients [4, 5]. During the same period, genetically engineered T cell receptors (TCR) were explored to achieve robust T cell responses which ultimately led to the emergence of chimeric antigen receptor (CAR) T-cell therapy [6, 7]. In 2017, CD19-CAR (tisagenlecleucel) became the first FDA-approved CAR-T cell therapy for treating patients with relapsed or refractory B cell acute lymphoblastic leukemia (ALL) [8].

The first ACT-based attempts for GBM therapy started about 40 years ago when several case reports showed that autologous leukocyte infusion into the resection cavity at the time of tumor resection may improve patient’s survival without toxicity [9‐11]. However, these approaches lacked specificity. To overcome this, genetic engineered T cells have been generated to specifically target brain tumor associated antigens (TAA) and have shown promising pre-clinical results. For GBM, interleukin-13 receptor alpha 2 (IL13Rα2) became the first CAR T-cell target tested in the clinic for its specific overexpression in tumors but not in normal tissues [12]. Epidermal growth factor receptor variant III (EGFRvIII) [13], human epidermal growth factor 2 (HER2) [14] and erythropoietin-producing hepatocellular carcinoma A2 (EphA2) [15] were also developed as major targets for their overexpression in GBM but not in healthy brain tissues. Other newly developed targets include ganglioside 2 (GD2) [16, 17], B7-H3 [18] and chlorotoxin [19]. Although all these targets have shown promising preclinical results and are evaluated in clinical trials (see Table 1 for summary), lack of clinical efficacy indicates that genetic modifications and therapeutic combinations are required in order to achieve a better response. In this review, we will introduce the basic CAR structures and their optimizations, the major targets that are in pre-clinical and clinical development for targeting GBM, and the combination strategies to improve CAR T-cell efficacy.

The tumor microenvironment is orchestrated by immunosuppressive cytokines, regulatory modulators and co-inhibitory receptors that regulate the responsiveness of immune cells [86]. While active CAR T cells elicit specific recognition and killing activities against tumor cells, the chronic exposure to tumor cells results in T cell exhaustion mediated by immune checkpoint pathway activation that is initiated by tumor cells, leading to a reduced ability to proliferate, produce cytokines and attack the tumor (Fig. 1c). Exhausted T cells upregulate immune inhibitory receptors, such as programmed death ligand-1 (PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Blocking this immune suppressive signaling has led to the development of ICIs such as nivolumab and durvalumab (PD-1/PD-L1 inhibitors) and ipilimumab (CTLA-4 inhibitors). These ICIs are FDA-approved for treating several types of cancer including melanoma, hepatocellular carcinoma and lymphoma and have shown significant clinical results [87]. Since ICIs eliminate T cell exhaustion, combining their use may further enhance CAR T-cell therapy efficacy through extended T cell proliferation and sustained tumor-killing activity. A recent case report has shown that administration of the PD-1/PD-L1 inhibitor pembrolizumab after CD19-specific CAR T-cell therapy in refractory diffuse large B-cell lymphoma induced a clinically significant antitumor response, suggesting that the PD-1 pathway may be critical in determining the response to CAR-modified T-cell immunotherapy [88]. However, despite the promising clinical results in other types of cancer, the use of ICIs in GBM clinical trials remain controversial. Nivolumab, a PD-1/PD-L1 inhibitor, failed to prolong overall survival of patients with recurrent GBM, leading to a discussion of whether ICIs may benefit GBM patients after all [89‐91]. Nevertheless, a recent trial with 35 patients with recurrent, surgically-removable GBM showed that patients who receive neoadjuvant PD-1 blockade, with continued adjuvant therapy following surgery had significantly better overall survival compared to those who received adjuvant or post-surgical PD-1 blockade alone [92]. Another study also suggests that combination of local chemotherapy with PD-1 blockade enhanced antigen-specific T effector cell expansion and improves survival in GBM models [93]. These studies suggest that anti-PD-1 blockade alone may not directly benefit GBM patients but may improve the efficacy of other therapeutics when used in combination. Currently, clinical trials are on-going to evaluate the therapeutic efficacy of EGFRvIII-CAR (NCT03726515) and IL13Rα2-CAR (NCT04003649) T-cell therapy in combination with ICIs such as pembrolizumab or nivolumab in recurrent or refractory GBM patients (Table 1). We anticipate that anti-PD1 blockage may improve the CAR T-cell efficacy for treating GBM patients.

Over the past decades, we have learned key principles to manage CAR T-cell efficacy: the specificity of the targeted antigen, the sufficient TCR activation by the specific antigen binding domain, the level of T cell activation and longevity, and the hostile micro-environment for T cells to penetrate, which also serves a source of immunosuppressive factors [94]. While many types of CAR T cells are now in clinical trials (Table 1), further optimization with advanced approaches are expected to improve their overall clinical efficacy.

Since genetic modification of CAR vectors is showing promising results in improving CAR T-cell efficacy, gene editing of the T cell is now a new strategy to further improve CAR T cells. Recently, the CRISPR/Cas9 mediated gene editing system has been applied to delete or mutate checkpoint-associated genes in CAR T cells, and has shown improvement in T cell persistence and survival [95‐97]. Using CRISPR/Cas 9-mediated deletion, Ren et al. [95] generated CD19-CAR T cells simultaneously deficient in TCR, HLA class I molecule and PD1, which showed more potent antitumor activities than non-edited CAR T cells when tested in xenograft mouse models. Notably, the TCR and HLA class I double-deficient T cells further reduced MHC restriction and eliminated alloreactivity and potential graft-versus-host disease (GVHD), thus making the strategy particularly useful for treating different patients. A different study using CRISPR/Cas9-mediated deletion of TCR α subunit constant (TRAC) region, beta-2 microglobulin (B2M) and PD-1 also showed profound anti-tumor activity in vivo [97]. Recently, Li et al. [98] investigated the approaches to improve CAR T transduction efficacy and CRISPR/Cas9 mediated PD-1 deletion using a 2-in-1 lentivirus vector and found that blocking anti-viral signaling in human primary T cells enhanced lentiviral-mediated transduction efficacy of a combinatory CAR/CRISPR vector for targeting HER2 and inhibiting PD-1 at the same time. Most importantly, the first phase 1 clinical trial using CRISPR/Cas9 for multiplex gene editing on T cells (NCT03399448) has demonstrated safety and feasibility in three patients with refractory cancer [99]. Given that the CRISPR/Cas9 approach has been widely applied to functional gene screening and validation, it also opens a new avenue to discover and target novel modulators of immune suppression. Through CRISPR/Cas9-mutagenesis screening, Wei et al. [100] have identified Regnase-1 as a major negative regulator of anti-tumor responses in CD8⁺ T cells, and that CD19-CAR T cells with deleted Regnase-1 showed greater longevity and a more vigorous anti-tumor response in B16 ovarian and B6 melanoma mouse models. We anticipate that future GBM-specific studies also will explore these novel ideas and approaches, which hopefully will further enhance CAR T-cell proliferation, longevity and long-term anti-tumor activity.

Brain tumors are considered one of the “most-difficult-to- treat” solid tumors. With the most recent advances in immunotherapy, the CAR T-cell therapy has become a revolutionary approach for treating hematological malignancies and it has great potential for brain tumors. This review intends to introduce interpretations of the most relevant papers addressing the use of CAR T cells for the treatment of GBM. We discussed the CAR designs and optimization, the major CAR T cell targets in clinical trials, as well as the strategies developed to improve CAR-T cell efficacy in the context of GBM. We anticipate that future clinical trial designs will not only focus on efficacy and safety, but also on the mechanisms involved in the immune response and resistance, and that the next generation of CAR T cell therapy will become an effective and safe therapeutics for treating malignant GBM.