Challenges in the Treatment of Glioblastoma by Chimeric Antigen Receptor T-Cell Immunotherapy and Possible Solutions

Abstract

Glioblastoma (GBM), one of the most lethal brain cancers in adults, accounts for 48.6% of all malignant primary CNS tumors diagnosed each year. The 5-year survival rate of GBM patients remains less than 10% even after they receive the standard-of-care treatment, including maximal safe resection, adjuvant radiation, and chemotherapy with temozolomide. Therefore, new therapeutic modalities are urgently needed for this deadly cancer. The last decade has witnessed great advances in chimeric antigen receptor T (CAR-T) cell immunotherapy for the treatment of hematological malignancies. Up to now, the US FDA has approved six CAR-T cell products in treating hematopoietic cancers including B-cell acute lymphoblastic leukemia, lymphoma, and multiple myeloma. Meanwhile, the number of clinical trials on CAR-T cell has increased significantly, with more than 80% from China and the United States. With its achievements in liquid cancers, the clinical efficacy of CAR-T cell therapy has also been explored in a variety of solid malignancies that include GBMs. However, attempts to expand CAR-T cell immunotherapy in GBMs have not yet presented promising results in hematopoietic malignancies. Like other solid tumors, CAR-T cell therapies against GBM still face several challenges, such as tumor heterogeneity, tumor immunosuppressive microenvironment, and CAR-T cell persistence. Hence, developing strategies to overcome these challenges will be necessary to accelerate the transition of CAR-T cell immunotherapy against GBMs from bench to bedside.

Keywords: CAR-T; adoptive immunotherapy; cellular immunotherapy; chimeric antigen receptor; glioblastoma.

Figure 1

The main course of developing CAR-T cell therapies on GBMs.

Figure 2

CAR designs to overcome the GBM intratumor heterogeneity. (A) CAR targeting multiple antigens: (1) Bispecific CAR (BiCAR, middle): BiCAR-T cells co-express two CARs that target different antigens on tumor cells. (2) Tandem CAR (Tan-CAR, right): Tan-CAR joins two antigen-binding domains to make a tandem CAR exodomain that can be activated by encountering either or both different antigens, e.g., HER2 and IL13Rα2. (3) Bispecific T-cell Engagers (BiTEs) secretory CAR (left): BiTEs are composed of two distinct arms: one arm targeting the wild-type EGFR on tumor cells and another arm specifically binding to the CD3ϵ subunit on endogenous T cells. BiTEs-CAR T cells can directly kill tumor cells that express EGFR-vIII, while indirectly redirecting endogenous T cells to eliminate tumor cells expressing wild-type EGFR through secreting BiTEs. (B) Logic-gate principle: (1) “AND” gate (left): the intracellular domain of one CAR is designed as a synNotch receptor structure that is cleaved to form a transcription factor after engaging antigen 1 and subsequently initiate the expression of another CAR specific for antigen 2. Thus, the tumor-killing effect can only be achieved when the CAR-T cells encounter tumors cells simultaneously expressing both antigen 1 and 2. (2) “NOT” gate (middle): similar to the “AND” gate design, except that activation of one CAR (targeting antigen 2) leads to suppressing the activation of another CAR (targeting antigen 1). Therefore, these CAR-T cells can be activated only if they encounter antigen 1 without the presence of antigen 2. (3) “AND+OR” gate (right): the first CAR is designed as in the “AND” gate design, and the following expressed CAR is a TanCAR structure (“OR” gate), which can be activated when it encounters antigen 1 or 3. CAR, chimeric antigen receptor; TCR, T-cell receptor; scFv, single-chain variable fragment; TF, transcription factor; tBID, truncated BH3 interacting death agonist.

Figure 3

Strategies to overcome the highly immunosuppressive GBM microenvironment. (A) The immunosuppressive microenvironment can limit CAR-T cell functions through several ways: ① upregulating PD-1 and other immune checkpoint molecules; ② increasing IL-4 and IL-13 that promote the transformation of TAMs into anti-inflammatory phenotype; ③ existence of GSCs that directly and indirectly (through activating Treg cells and M2-type TAMs) inhibit CAR-T cell functions; ④ increasing the infiltration of Treg cells that directly and indirectly (through secreting IL-10, TGF-β, etc.) suppresses CAR-T cell functions. (B) The corresponding strategies in CAR designs to block or reverse these immunoinhibitory effects. ① Three methods for blocking the PD-1 pathway: combining with antibodies blocking the PD-1 molecule (left), knocking out the PDCD1 gene (encoding PD-1) by the genome editing method (middle), and using a PD-1 chimeric switch receptor (right) that reverses the inhibitory signal by PD-1 activation into the stimulatory signal within CAR-T cells; ② CAR-T cells armed with IL-8 receptor (CXCR1 and CXCR2) could be attracted into tumors enriched with IL-8 and neutralized its immune-inhibitory effect; ③ the fourth-generation CAR-T cells armed to secrete proinflammatory cytokines that can enhance the direct tumor-killing activities by CAR-T cells as well as the indirect bystander killing by endogenous T cells; ④ inverting the inhibitory effects of anti-inflammatory cytokines through transgenic expression of an inverted cytokine receptor that fuses the IL-4 receptor exodomain with the IL-7 receptor endodomain that activates T cells. MDM, myeloid-derived macrophages; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; GSC, glioma stem cell.

Figure 4

Strategies to prolong CAR-T cell persistence. (A) Tuning inherent signaling. High phosphorylation intensity in TCR signaling leads to robust but unsustainable antitumor activity, while alleviating the phosphorylation intensity results in reduced but sustained tumor-killing effects. Two strategies can be utilized in CAR design to tune the phosphorylation intensity, thereby prolonging CAR-T cell persistence while maintaining moderate antitumor activity: ① Integrating CD28-CARs with the FRB domain, which recruits phosphatases via binding FKBP and then decreases the phosphorylation level, can suppress CAR overactivation. ② Intermittently administering a small molecular drug, shield-1, to interrupt the dissociation effect on CARs by the DD domain that is fused with CARs, will block CAR continuous activation. (B) Screening pro-exhaustion genes: GBM cells and their microenvironment can adaptively alter the gene expression profiles of infiltrative T cells into an exhausted phenotype, leading to shortened persistence. Identification of these pro-exhaustion genes via CRISPR-based genome-scale knockout technology will greatly accelerate CAR-T cell development to prolong persistence. LCK, leukocyte-specific protein tyrosine kinase; FKBP, FK506 binding protein; FRB, FKBP-rapamycin binding; DD, destabilizing domain.