Bottlenecks and opportunities in immunotherapy for glioma: a narrative review : Journal of Bio-X Research

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Bottlenecks and opportunities in immunotherapy for glioma: a narrative review

Shi, Yinga; Wu, Mengwana; Liu, Yuyangb; Chen, Lingb; Bian, Xiuwuc,*; Xu, Chuana,d,*

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Journal of Bio-XResearch 5(4):p 151-162, December 2022. | DOI: 10.1097/JBR.0000000000000135

Abstract

Introduction

Gliomas are the most common primary malignant tumors of the central nervous system (CNS) that originate from glial cells. They rank among the top four most lethal malignant tumors, with an incidence of 4.67 to 5.73 per 100,000 persons.[1] The fifth edition of the World Health Organization classification of central nervous system tumors (WHO CNS 5) classifies gliomas into diffuse gliomas, astrocytomas, oligodendrogliomas, and glioblastomas based on their histological features. More accurate molecular phenotypes are also mentioned, including those with mutations of isocitrate dehydrogenases (IDH)-1/2 and 1p/19q co-deletion.[2] Surgical removal of tumor tissue followed by radiotherapy and/or chemotherapy (including temozolomide [TMZ], lomustine, intravenous carmustine, carmustine wafer implants, and bevacizumab) has remained the standard therapy for gliomas.[3] However, the median survival time of patients who undergo standard therapy is only 14.6 months; this may be is attributed to tumoral heterogeneity and limited access to current medicines.[4]

Since the last century, innovations in immunotherapy have attempted to address the dilemmas in the treatment of gliomas. The brain is an immune privileged organ, with lower lymphocyte quantity and activity. The immunosuppressive intracranial microenvironment is regulated by abundant cell types and their products, such as vascular cells, microglia, neural precursor cells, and peripheral immune cells.[5] Three main obstacles impede adequate mobilization of effective immune cells (Fig. 1). The first is the physical barrier, namely, the blood-brain barrier (BBB), which is formed by endothelial and mural cells. The tight junctional construction of the BBB supports maintenance of central nervous system (CNS) homeostasis, separating the blood components, pathogens, and circulating immune cells in the CNS from the peripheral environment.[6] The second impediment is reduced tumor immunogenicity, which is commonly found in almost all solid tumors. A low tumor mutation burden or the absence of antigen presentation machinery is responsible for the paucity of effective antigen targets; this conceals tumor characteristics, making it invisible to the immune system.[7] T cell dysfunction is the third hindrance; effective T cells are exhausted by persistent activation, which is manifested by the loss of cytotoxic products including interleukin-2 and interferon (IFN)-γ. Therapeutic options including radiation or TMZ have also been reported to reduce lymphocyte count; this is associated with shorter survival time in patients with glioma.[8] Therefore, switching of the immune environment from “cold” to “hot” status is a prerequisite for overcoming the state of immune silence.

F1
Figure 1.:
Immunosuppressive environment of glioma. There are three main obstacles contributing to immune suppression in glioma: existence of blood-brain barrier prevents infiltration of immune cell into intracranial position; low tumor mutation burden or the absence of antigen presentation machinery are responsible for reduced tumor immunogenicity; infiltrated T cell become dysfunctional under persistent activation. Created with BioRender.com. CTL=cytotoxic T lymphocyte, IFN-γ=interferon gamma, IL-2=interleukin-2.

Various therapeutic strategies targeting different aspects of immunosuppression have been proposed to address this issue; their clinical potential is being fully evaluated (Fig. 2). Tumor vaccines have been utilized to address the low immunogenicity of gliomas; they supplement immunogenic neoantigens or enhance antigen presentation (in the case of dendritic cell [DC] vaccines). Adoptive cell therapies such as chimeric antigen receptor (CAR)-T or CAR-natural killer (NK) cells are designed to address the issue of inadequate immune cell infiltration, while inhibitors targeting immune checkpoints are used to recover functionally exhausted T cells. Although high response rates are achieved in almost all tumors, outcomes remain unsatisfactory in patients who undergo immunotherapy. It is believed that this may be overcome by combining immunotherapy with standard therapy or by other innovative strategies such as tumor-treating fields (TTFields). In this review, we illustrate the principles of current immunotherapies and related combined therapies in patients with glioma. Updated information regarding the advances and limitations of these therapies has been summarized after a thorough search of related clinical trials. Further challenges in the development of immunotherapy have also been discussed.

F2
Figure 2.:
Landscape of immune cells and components in glioma. Glioma microenvironment is composed of highly heterogeneous tumor cells and various immune cells, such as macrophage and lymphocytes. Under different stimulation or pathological condition, these cells play different immune modulatory function by expressing distinct markers or releasing cytokines. Created with BioRender.com. AIM2=absent in melanoma, CCL17=C-C Motif Chemokine Ligand 17, CCL20=C-C Motif Chemokine Ligand 20, CCL22=C-C Motif Chemokine Ligand 22, CTLA-4=cytotoxic T lymphocyte-associated antigen 4, DC=dendritic cell, DC-SIGN=dendritic cell-specific ICAM-3-grabbing nonintegrin, EGFRvIII=epidermal growth factor receptor variant III, EphA2=ephrin type-A receptor 2, FN=fibronectin, GARP=glycoprotein-A repetitions predominant, gp100=glycoprotein 100, HA=hyaluronic acid, HER2=human epidermal growth factor receptor 2, HLA-E=HLA class I histocompatibility antigen, alpha chain E, IDH1=isocitrate dehydrogenase (NADP(+)) 1, IDO1=indoleamine 2,3-Dioxygenase 1, IFN-γ=interferon gamma, IL-10=interleukin 10, IL13Rα2=interleukin 13 receptor α2, IL-2=interleukin 2, IL-4/13=interleukin 4/13, IL-6=interleukin 6, ITGB1=integrin subunit beta 1, ITGB2=integrin subunit beta 2, LOXL2=lysyl oxidase like 2, Mac-1=macrophage-1 antigen, MAGE-A1=MAGE family member A1, MHC I/II=major histocompatibility complex I/II, MICA=MHC class I polypeptide–related sequence A, NCR=natural cytotoxicity receptor, NCR-L=natural cytotoxicity receptor-ligand, NK=natural killer, NKG2A=NK cell Group 2 isoform A, NKG2C=NK cell Group 2 isoform C, NKG2D=natural killer group 2, member D, PD-1=programmed cell death 1, PGE2=prostaglandin E2, Qa-1=MHC class Ib molecule, SOX11=SRY-Box transcription factor 11, SOX2=SRY-Box transcription factor 2, TGF-β=transforming growth factor beta, Th1=T helper type 1, Th2=T helper type 2, TNF-α=tumor necrosis factor α, Treg=regulatory T cell, TRP2=tyrosinase-related protein 2, VACN=versican, WT1=Wilms tumor 1.

Retrieval strategy

Literature review was electronically performed using PubMed database. The following combinations of key words were used for initial literature retrieval: glioma and immunotherapy; neoantigen; DC vaccine; immune checkpoint inhibitor; chimeric antigen receptor-T; tumor-treating fields. Most of the selected studies (80% of all references) were published form 2012 to 2022. An ancient publication (1991) was included in consideration to its relevance in the mast cell field. Information of clinical trials included in this review was retrieved in ClinicalTrial.gov (https://clinicaltrials.gov).

Tumor vaccines

The genomes of cancer cells contain nonsynonymous somatic mutations, which are entirely absent in their healthy counterparts. These mutation-derived immunogenic neo-nonself epitopes (neoantigens) can be presented by major histocompatibility complexes (MHCs) to distinct T cell receptors (TCRs) to trigger an antitumor T cell response. Patients with a higher tumor mutation burden tend to be capable of producing neoantigens more frequently; this becomes a prognostic factor for immunotherapeutic response.[9] However, silenced expression of effective tumor antigens is a universal phenomenon in gliomas. These altered antigens are potentially labeled as mismatched production and are cleaved by protease or metalloproteinase systems; this deprives their access to MHC molecules. Gliomas also evolve to reduce MHC diversity by loss of major MHC encoding-components (eg, human leukocyte antigen loss of heterozygosity) or epigenetic regulation; this minimizes their immunogenicity.[10] Advancements in antigen discovery and vaccine development have led to the use of multiple vaccine approaches aimed at supplementing immunogenic tumor antigens; (pre-) clinical assessments are ongoing (Additional Table 1, https://links.lww.com/JR9/A43).

Single neoantigen vaccines

Ideal neoantigens, which can either be distinct mutated proteins or related nucleic elements, are expected to elicit sufficient tumor-specific immune responses. Cancer-specific mutant antigens are the leading type of tumor vaccine material; they are generally peptide(s) (usually 10–30 amino acids long) that are encoded by candidate neoantigen-encoding genes.[11] The detailed landscape of glioma-specific antigens has been expanded using sequencing technology and computational algorithms; the antigens include epidermal growth factor receptor variant III (EGFRvIII), IDH-1/2, interleukin-13 receptor (IL13R) α2, and survivin.[12]

Mutations of the EGFR gene are distinctive of glioblastoma multiforme (GBM) and multiple tumors and are accompanied by aberrant activation of the EGFR cascade. EGFRvIII is the most common variant of EGFR (deletion of exons 2–7) found in GBM, with 31% of GBMs overexpressing both wildtype (wt)-EGFR and EGFRvIII.[13] Tumoral specificity and membrane localization of EGFRvIII makes it a practicable target for antibody-based drugs, such as depatuxizumab mafodotin (Depatux-M) and AMG595. Rindopepimut (also known as CDX-110), a vaccine consisting of an EGFRvIII-specific peptide conjugated to keyhole limpet hemocyanin, has shown good tolerance and immunogenicity in patients with GBM. However, rindopepimut offers limited clinical benefit.[14] Loss of EGFRvIII expression is prevalent in patients who experience recurrence following treatment with rindopepimut; EGFRvIII-free cells may be responsible for resistance to the drug.[15]

Mutations on critical arginine residues of IDH (R132H) are rarely found in healthy cells; however, these are enriched in gliomas, which produce the oncometabolite 2-hydroxyglutarate and demonstrate genomic hypermethylation.[16] In a first-in-humans phase I trial (NCT02454634), peptide vaccines using mutated IDH demonstrated successful presentation by MHC II; this induced mutation-specific T cell responses with interferon-γ production.[17] The lysine (K) to methionine (M) mutation at position 27 of histone H3 (H3.3K27M) is another characteristic of gliomas and is harbored by approximately 70% of these patients. H3.3K27M-targeted peptide vaccines show immunogenicity and good tolerance in patients with GBM.[18] Patients with H3.3K27M-specific immunological CD8+ T cell responses demonstrate prolonged overall survival (OS) compared with non-responders.[19]

Mixed antigen vaccines

Compared to single-neoantigen cancer vaccines, mixed antigen peptide vaccines demonstrate greater antitumor efficacy. IMA950 is a novel GBM-specific therapeutic vaccine, which contains 11 tumor-associated peptides with high binding affinity for human leukocyte antigen (HLA).[20] Injected IMA950 can be presented on tumor surfaces, eliciting spontaneous T cell responses in a majority of patients with GBM.[21] In patients with astrocytoma, sustained CD8+ and CD4+ T cell responses have been found to appear after vaccination with IMA950 and adjuvant polyinosinic-polycytidylic acid.[22] Aborted loading of tumor epitopes to HLA receptors lead to unfavorable responses to exogenous tumor-associated antigen (TAA) vaccines. This issue may be addressed by personalizing neoantigens; this may be achieved by removal of identifiable tumor-associated peptides using endogenous antigen-processors.[23] In the notable glioma actively personalized vaccine consortium-101 trial (NCT02149225), vaccination strategies of mutated peptides derived from the GBM library (APVAC1) or personalized mutated neoepitopes (APVAC2) were assessed to be safe and functional, with sustained immunogenic T cell responses in patients treated with APVAC1 or APVAC2 neoepitopes.[24] In another phase Ib trial (NCT02287428), neoantigens were predicted and designed by comparing surgically resected tumors and matched normal cells. After vaccination with those personalized peptides, a subset of T cells was induced within resected GBM tumors.[25] In addition to those protein antigens, particular antigen-encoding deoxyribonucleic acid (DNA) or ribonucleic acid were selectively taken up by resident antigen processing cells (APCs) to allow efficient expansion and activation of antigen-specific T cells.[26] In this context, the intraperitoneally injected SOX6 DNA vaccine has successfully induced SOX6-specific cytotoxic T lymphocyte (CTL) responses, effectively restricting tumor growth.[27]

Oncolytic virus-based vaccines

Oncolytic viruses represent a new class of immunotherapy for reviving tumor immunogenicity, which is dually accomplished by specifically recognizing and infecting tumor cells.[28] The viral contents effectively recruit immune cells, while tumor cells are lysed during the release of viral progeny; massive TAAs are released to serve as “in situ vaccines,” attracting nearby or distal immune cells and inducing a systemic immune response. Human herpes simplex virus (eg, HSV-1 C134, HSV-1 rQNestin34.5v.2, HSV-1 G207, and HSV-1 M032), adenovirus (eg, DNX-2401, DNX-2440, and CRAd8-S-pk7-loaded neural stem cells), and parvovirus (eg, ParvOryx) are the leading types of oncolytic viruses being engineered for virotherapy. Present evidence suggests that their use is feasible and safe, with minimal dose-limiting toxic effects or serious adverse events.[29,30]

DC vaccines

APC-mediated tumoral antigen processing and uptake is necessary for initiation of T cell responses. This is implemented by abundant antigen processing and presenting molecules (eg, MHC), co-stimulatory factors (eg, CD80, CD86, and CD40), and adhesion molecules (eg, intercellular adhesion molecule 1 and lymphocyte function-associated antigen 1). DCs normally serve as the most potent APC population in different cancers; however, the quantity or capacity of DC antigen uptake is impaired in glioma.[31] This phenomenon is associated with TMZ treatment, indicating the feasibility of DC vaccine use in patients with glioma receiving TMZ.[32] Direct supplementation of DC may address this dilemma through sensitization of antigen-reacting T cells.

Notably, immature DCs need to be educated to recognize specific tumor antigens before injection. Both, general glioma-associated antigen peptides and personalized autologous tumor lysate (ATL) can be used to pulse DCs and recover their tumoricidal activity. Similar to neoantigens, satisfactory antigens are more glioma-specific and encode HLA-restricted T cell antigen epitopes. Interleukin 13 receptor α2 (IL13Rα2) is a glioma-associated antigen, which can offer better induction of specific and tumor-reactive CTLs.[33] DC pulsed with an IL13Rα2-derived HLA-A*2402-restricted peptide, WYEGLDHAL (the peptide sequence), enables positive CTL responses in patients with recurrent gliomas.[34] In a phase I clinical trial, the injection of peptide cocktail (Wilms tumor-1, human epidermal growth factor receptor 2 [HER2], melanoma-associated antigen 3, and melanoma-associated antigen 1 or gp100) pulsed-DCs contributed to a positive immunological response and long-term recurrence-free survival in GBM.[35] In a subsequent phase II clinical trial, positive CTL responses were elicited in most patients, and were accompanied by prolonged survival.[36] Certain pathogenic antigens manifest particularly valuable immunogenic ability. The human cytomegalovirus (HCMV) is a carcinogenic virus, which infects nearly half of the adult population; HCMV antigens or nucleic acids have been preferentially found in GBM cells rather than in adjacent healthy tissue. Vaccination with DCs loaded with GBM lysate therefore elicits a HCMV-specific immune response.[37]

Compared to glioma-associated antigen-DC vaccination, ATL-DC vaccination is more precise and well-tolerated. Autologous glioma cells are irradiated to release tumor antigens, which are fused with DCs in distinct ratios and conditionally cultured before administration. Integrating autologous DC vaccines with radio/chemo/immunotherapy can potentiate their anti-tumor activity.[38] DCVax-L (Northwest Biotherapeutics, Inc., Bethesda, MD) is one of the earliest ATL-DC vaccines used in glioma. Results from a phase 3 trial demonstrated the safety and efficacy of adding DCVax-L to standard therapy; it also extended survival duration to a certain extent.[39] Combining DCVax-L with a neoantigen-based peptide vaccine even prolonged OS to 21 months in 1 patient with GBM.[40]

The function of DC vaccines may be hindered by distinct immune cell subsets and cytokines, such as IL-10, that lead to ineffective T cell responses. In patients with glioma who receive DC vaccines, elevated T regulatory (Treg) cell counts and expression of negative co-stimulatory molecules are associated with poor prognosis.[41] Decreased post/pre-vaccination Treg, to activated NK cell ratios are associated with prolonged survival.[42] Administration of immune stimulators such as polyinosinic-polycytidylic acid or tetanus toxoid enhances the effectiveness of DC vaccines, promotes tumor antigen presentation, and significantly improves survival in patients with GBM.[43]

Immune checkpoint blockade

Once an MHC presented tumor antigen is recognized by TCRs (ie, the first signal), a second signal is launched on binding of T cell co-stimulatory receptors with corresponding ligands on APCs. CD28 represents the major co-stimulator in almost all solid tumors, playing a crucial role in regulation of T cell activation and sensitivity.[44] Using the MYPPPY motif within its extracellular immunoglobulin (Ig)-V-like domain, CD28 can combine with members of the B7 family, which are highly expressed on APCs; these include B7-1 (CD80) and B7-2 (CD86). Once CD28 engages with CD80 and CD86, nearby membrane lipid rafts are rearranged to augment TCR signaling and cytokine production. In addition to the co-stimulatory process, a set of co-inhibitory factors can competitively bind with similar homo-ligands to functionally exhaust T cells and impair tumoricidal ability.[45] These co-inhibitory factors target different mediators of the exhaustion process, such as programmed death 1 (PD-1/B7-H1/CD279), PD-L1 (B7-H4/CD274), PD-L2 (B7-DC/CD273), CD276 (B7-H3), cytotoxic T-lymphocyte–associated antigen 4 (CTLA4 [CD152]), lymphocyte-activation gene 3, T cell immunoglobulin and mucin-domain containing-3 (HAVCR2/CD366), and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, among others.[46]

PD-1, in conjunction with its ligands (PD-L1 or PD-L2), are the most well-known co-inhibitory couples in various tumors. PD-1 is commonly enriched in myeloid lineage cells such as macrophages and dendritic cells, while PD-L1 is abundantly found in glioma and microglial cells.[47] After combining with PD-L1 or PD-L2, PD-1 is phosphorylated at the tyrosine-based inhibitory motifs, providing docking sites for Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2. Activation of SHP-1 and SHP-2 dephosphorylates the TCR–CD3ζ complex, blocking proximal CD28 co-stimulation and TCR signaling.[48] PD-1 or PD-L1-targeted monoclonal antibodies have been previously used to counteract their immune-inhibitory effect; some of these are already clinically approved by the United States Food and Drug Administration (FDA). Nivolumab (Opdivo) and pembrolizumab (Keytruda) are the most well investigated PD-1 antibodies. The safety and feasibility of nivolumab therapy has been demonstrated in patients with relapsed GBM (NCT02550249).[49] Patients receiving neoadjuvant pembrolizumab (which is continued in the adjuvant setting) have also demonstrated significantly longer OS than those receiving adjuvant PD-1 alone.[50] As with other immunotherapy agents, response to anti-PD-1 inhibitors is highly associated with basic immunological status and tumor conditions, such as hyper tumor mutation burden.[51]

CTLA-4 is another key co-inhibitor, which combines directly with CD3-ζ to inhibit tyrosine phosphorylation-mediated T cell activation. CTLA-4 can mediate trans-endocytosis and degrade CD80 and CD86 from opposing cells, irretrievably terminating co-stimulation of CD28.[52] Blocking CTLA-4 with specific antibodies therefore reactivates T cells and improves the anti-tumor response.[53] Previous literature has demonstrated that CTLA-4 and PD-1 act separately and in an orderly manner to inhibit T cell activation or drive T cell exhaustion; this is suggestive of the synergistic antitumor effect of combined anti-CTLA-4 and anti-PD-1 therapy.[54] This was validated by the findings of the CheckMate (name of the clinical trial series)-143 study, in which patients with recurrent GBM were administered nivolumab plus ipilimumab, the first humanized anti-CTLA-4 monoclonal antibody.[55] CTLA-4 blockade also restricts disease progression and leads to acquisition of immune responses in patients with brain metastases; the intracranial response is more prolonged with combined nivolumab and ipilimumab therapy than with either agent alone.[56]

Although immune checkpoint inhibitors (ICIs) are widely used in other cancers, gliomas demonstrate higher tolerance, which may be attributed to insufficient immune cell infiltration.[57] Expression of immune checkpoints and T-cell hypo-responsiveness are prominently upregulated in glioma; this results in more severe immune exhaustion than in other malignancies.[58] The dynamic process of T cell activity interference is mediated by numerous immune checkpoint molecules, which render the exhaustion process reversible in early stages; however, it progresses rapidly in later phases due to dysfunction.[59] PD-1 promotes cell proliferation and self-renewal in glioma stem cells without participation of PD-L1; therapeutic antibodies that inhibit PD-1/PD-L1 interactions therefore fail to diminish the growth advantage of PD-1 in tumor cells.[60] The identification of new immune checkpoint molecules may provide a solution. An increasing number of immune checkpoint molecules are being explored and evaluated by ongoing clinical trials, such as those on T cell immunoglobulin and mucin-domain containing-3 and lymphocyte-activation gene 3. In a study, multitarget combined therapy (eg, PD-1, CTLA-4, and B and T lymphocyte attenuator-monoclonal antibodies) synergistically increased the effective cure rate in GBM; it also markedly improved systematic immunologic memory and prevented recurrence.[61] Notably, anti-antibody reactions to these animal-derived antibodies and treatment-related adverse events are additional challenges encountered in ICI therapy.

Augmentation of tumor-killing lymphocytes

CAR-T cells

Dysfunction or loss of CD8+ T cells is a universal phenomenon in glioma, and is more common in the tumor core than the peritumoral zones.[62] The major objective of current immunotherapy is to prime the response of tumor-killing lymphocytes; this can be achieved by adoptive cell therapy (ACT). Two types of cells are usually employed to reinforce defective CD8+ T cells in ACT: tumor-specific CTLs and nonspecific NK cells. CAR-T therapy supplements CTLs by transfusion of autologous or xenogenous T cells loaded with specific TAA-recognition TCRs. The fourth-generation of CAR-Ts are armed with co-stimulatory molecules and suicide genes, and are demonstrating good safety and specificity in current clinical studies.[63]

The essential steps of CAR-T preparation include the design of single-chain variable fragments of cell-surface receptors and loading of the coding sequence onto T cells for expression of engineered TAA-specific receptors. As described previously, TAAs can be classified into unique or shared tumor antigen categories based on their varied distribution in normal or neoplastic tissues.[64] Similar to neoantigens, an ideal TAA selected for CAR-T construction must be detectable and tumor-specific. CAR-Ts targeting IL13Rα2 (NCT2208362), CD276 (NCT04385173 and NCT04077866), HER2 (NCT03500991), ephrin type-A receptor 2 (EphA2) (NCT03423992), and disialoganglioside 2 (NCT04196413), among others, are currently under evaluation in glioma.[65]

As mentioned previously, EGFRvIII amplification occurs in ~50% of patients with GBM; this makes it a promising target for CAR-T therapy. In the NCT02209376 study, patients with GBM demonstrated detectable transient expansion of CAR-EGFRvIII T cells in their peripheral blood after being injected with CAR-EGFRvIII T cells; the T cells were successfully trafficked across the BBB into active GBM sites.[66] However, inevitable immune escape or exhaustion is also observed in internal CAR-T cells. Trafficking of CART cells to regions of active GBM is rapidly followed by a decrease in antigen levels in distinct areas; this leads to off-target effects and tumor relapse. In patients who recur following IL13Rα2-redirected CAR-T therapy, the overall levels of IL13Rα2 in recurrent tumor tissues are lower compared to pre-therapeutic levels; this is especially observed adjacent to the injection site of CAR-T cells (as in the NCT00730613 study).[67] This issue has been partially addressed in a subsequent study by fusing a 4-1BB co-stimulatory domain to the EGFR-specific CAR, thereby avoiding tumor escape of EGFRvIII- tumor cells.[68]

CD276, a homologue of PD-L1, is expressed in a wide range of malignancies including GBM and neuroblastoma.[69] Increasing evidence suggests that CD276 negatively regulates T cell activation and proliferation and effector cytokine production in patients with glioma; CD276-redirected CAR-T cells therefore effectively control tumor growth.[70] Pathogenic antigens are also thought to compensate for immunosuppression and increase T cell responses based on their special immunogenicity. In a phase I clinical trial, the co-stimulatory effect of TAA and latent viral antigens were measured using HER2-specific CAR-modified virus-specific T cells. Autologous HER2-specific CAR-modified virus-specific T cell infusions were found to be safe and offered clinical benefit in patients with progressive GBM.[71] In this context, treatment with HCMV-specific ACT has been found to trigger evident CMV-specific T cell immunity; it is safe and improves OS in GBM.[72]

Multi-target strategies have been adopted to overcome the off-target effect. Bivalent CAR-T (HER-2 and IL13Rα2), trivalent CAR-T (HER-2, IL13Rα2, and EphA2), and bispecific-CAR-T (EGFR and EGFRvIII) significantly mitigate tumor antigen escape and overcome antigenic heterogeneity in GBM models. In this context, EGFR-directed bispecific T-cell engager technology is a dual-targeted platform engineered onto EGFRvIII-CAR T cells; it has been found to minimize immune escape in EGFRvIII/wtEGFR+ tumors.[73] In a trial, an innovative synthetic Notch (synNotch) CAR circuit was designed to recognize EGFRvIII and sequentially produce CAR targets on other antigens, including EphA2 and IL13Rα2. The SynNotch-based CAR-T approach elicited thorough but controlled tumor cell killing, and averted persistent immune exhaustion.[74]

In recent years, the fifth generation of CAR-T, namely universal CAR-T, is under investigation. Functional elements such as the interleukin-2 receptor, which allows Janus kinase/signal transducers and activators of transcription pathway activation in an antigen-dependent manner, are loaded to optimize the antigen-specificity and scalability of CAR-T cells.[75] Appropriate doses and techniques for CAR-T injection are under investigation for improving safety and persistent activity issues. Engineered CAR-T cells are usually derived from autologous peripheral blood mononuclear cells to protect patients from immune rejection. Lymphodepletion chemotherapy is initially used to eliminate competition from growth-promoting cytokines and remove immune suppressive cells (eg, Tregs or myeloid suppressive cells). Patients are then infused with CAR-T cells at doses of 1 × 107 to 108 cells per cycle by intravenous or intracranial injection, to lower the risks of cytokine release syndrome or organ toxicity, especially neurotoxicity.[76] Intraventricular injection is an effective strategy for directing CAR-T cells into intracranial regions. Active CTLs cloned from CAR-T have been detected after being administered directly into the resection cavity via an indwelling catheter.[67] However, this technique may result in insufficient CAR-T cell counts in extracranial regions, thereby impairing the ability to eradicate metastatic tumors.[73] Intravenous injection is more convenient and is associated with a reduced risk of complications, including increased intracranial pressure. Increasing evidence suggests that intravenously injected T cells can also cross the BBB and travel to the brain, addressing concerns regarding homing to glioma tissue.

NK-T cells represent a distinct lymphocyte subset, in which TCR and NK lineage markers are co-expressed. They may be classified into three types: type I (invariant NKT [iNKT]), type II (nonclassical NKT), and NKT-like cells. In anti-tumor immunity, iNKT cells express invariant Vα24-Jα18 TCRα and Vβ11 TCRβ chains, which can be activated by recognition of CD1d presented antigens such as α-galactosylceramide. Treating GBM cells with retinoic acid upregulates CD1d expression, assisting the induction of iNKT cell-mediated cytotoxicity.[77] Nevertheless, the potential of NKT-based immunotherapy remains unused due to limited clinical evidence.

CAR-NK cells

Although the anti-tumor ability of CAR-T cell therapy has been confirmed, several limitations persist; these include possible disease progression during preparation (which is time-consuming) and off-target effects. CAR-NK based therapy is regarded as a more secure alternative. CARs targeting tumor specific antigens can be loaded onto NK cells to exert considerable antitumor activity. Notably, the anti-tumor cytotoxic function of NK cells is MHC-independent. NK-cells may therefore be derived from healthy donors, reducing the likelihood of toxicities or cytokine release syndrome.[78]

CAR-NK cells targeting HER2 are currently under evaluation in patients with recurrent HER2-positive GBM (NCT03383978); no dose-limiting toxicities have been reported to date.[79] Several preclinical studies have also highlighted the antitumor activity of infused CAR-NK cells. In a study, intravenous infusion of EGFRvIII-directed YTSDAP12.CD3/CAR-NK cells with CXCR4 receptor overexpression inhibited tumor growth and prolonged survival in xenograft mouse models.[80] Bispecific CAR-NK cells targeting both wt- and mutated EGFR (dual-specific EGFR- and EGFRvIII-directed CD28.CD3ζ.CAR-NK-92) have been constructed to reduce antigen loss. The bispecific structure has been found to outperform monospecific EGFRvIII-directed CAR-NK in terms of survival prolongation and reduction of antigen escape.[81]

Immunotherapy-based combination therapies

Although immunotherapies have shown efficacy in almost all solid tumors, current data suggest that they are only effective in specific biomarker-identified subgroups of patients. As sufficient immune infiltration is a prerequisite of immunotherapy efficacy, combining immunotherapy with other immune-stimulating strategies may exert a synergistic effect (Additional Table 2, https://links.lww.com/JR9/A44).

Immunotherapy + standard therapy

Data from studies are increasingly demonstrating the association between anti-tumor therapy and immunogenic cell death. Radiotherapy and TMZ can affect immune cell infiltration in gliomas via three different mechanisms: increased expression of adhesion molecules, chemokine secretion, and changes in vascular structure. However, in the CheckMate-498/NCT02617589 and CheckMate-548/NCT02667587 studies, the addition of ICI to radiotherapy or TMZ did not provide superior efficacy compared to radiotherapy plus TMZ treatment in patients with GBM.[82,83] These findings also underline the importance of predictive biomarker-based patient stratification prior to administration of immunotherapy plus standard therapy. In this context, baseline tumor genomic or gut microbiotas such as Ruminococcus are reported to be associated with OS and response to treatment.

In many patients with cancer, adjuvant therapy aids in the elimination of residual cancer cells after standard treatment, effectively preventing metastasis and reducing recurrence risks. A number of clinical trials have demonstrated the clinical benefit of adjuvant (after surgery and radiotherapy) TMZ in patients with glioma.[84] In this context, an immune inhibitory environment can be formed after surgery, radiotherapy, or TMZ treatment, with an increase in immune-suppressive lymphocytes and genetic markers.[85] Gliomas recurring after surgery show significantly increased levels of PD-1 and PD-L1 compared to presurgical lesions; this reflects the abundance of anti-PD-1/PD-L1 targets in unresectable gliomas. Adjuvant immune strategies, such as adjuvant DC vaccines or CMV-specific T cells, are therefore expected to reduce the risks of operation-induced micro-metastases and postoperative recurrence.[72,86]

Neoadjuvant immunotherapy can also generate enhanced and sustained antitumor immune responses.[87] The immunoenhancing effect reduces tumor burden and reactivates systemic immunity, thereby improving survival over standard monotherapy.[49] In patients with glioma, neoadjuvant vaccination with GBM stem cell lysate upregulates the secretion of cytokines and chemokines, with an increase in both peripheral and glioma-infiltrated CD8+ T cells.[88] In this context, immune-related genes were found to be upregulated while proliferative genes were downregulated in glioma tissue from patients who received neoadjuvant PD-1. Neoadjuvant and continued adjuvant pembrolizumab therapy has been found to significantly extend OS compared to adjuvant PD-1 blockade alone; this indicates synergetic benefit with neoadjuvant immunotherapy and surgery.[50] However, before this becomes the standard of care in patients with glioma, the schedule needs to be optimized based on genetic subtypes and individual responses.

Immunotherapy A + immunotherapy B

The immune suppressive microenvironment in glioma is collectively influenced by multiple factors or immune related processes, which are difficult to resolve using mono-immunotherapy or therapies aimed at a single target. Combining different types of immunotherapeutic treatments may stimulate distinct immune compartments, thereby enhancing antitumor immunity and overcoming resistance. In a study presented at the American Society of Clinical Oncology annual meeting, 2022, T cell-enabling therapy including INO (synthetic DNA plasmid)-5401 (synthetic DNA plasmid encoding human telomerase reverse transcriptase, WT-1, and prostate specific membrane antigen) and INO-9012 (synthetic DNA plasmid encoding IL-12) was administered in combination with cemiplimab, an inhibitor of PD-1, in patients with newly diagnosed GBM. INO-5401 promoted infiltration of antigen-specific T cells in GBM, turning “cold” microenvironments “hot,” thereby generating synergistic effects with cemiplimab.[89] In another right-to-try program, a tumor vaccine (SITOIGANAP) administered in combination with cyclophosphamide/granulocyte-macrophage colony-stimulating factor/bevacizumab/nivolumab (or pembrolizumab) offered significant benefit. The OS in these patients was twice that of the average in patients with recurrent GBM; however, the toxicity was minimal compared to that with current therapy.[90]

Immunotherapy + antiangiogenic therapy

Hypoxia-associated neovascularization is the hallmark of almost all solid tumors, and supplies oxygen and nutrition for fast-growing and invasive tumor cells. Vascular endothelial growth factor (VEGF)-mediated vasculogenesis and angiogenesis are primarily responsible for tumor-vessel formation. The extreme hypoxic conditions found in glioma additionally stimulate other inter-related patterns of neovascularization such as vascular mimicry and GBM-endothelial cell transdifferentiation.[91] Current anti-angiogenic agents include antibodies or peptides that target potent angiogenic factors (eg, bevacizumab, aflibercept, and thalidomide) and tyrosine kinase inhibitors (eg, axitinib and sorafenib). Bevacizumab, a humanized monoclonal IgG1 VEGF antibody, has been approved by the FDA for the treatment of GBM.[92] Although patients with glioma show superior responses with bevacizumab than other anti-vascular therapy, durable tumor control is not achieved. Tolerance to bevacizumab partly results from vasotropic factors other than VEGF (including hepatocyte growth factor and fibroblast growth factor, among others); these factors reinitiate vascular formation when VEGF is blocked. The anti-angiogenic function of bevacizumab can be replenished by plerixafor, a small molecular inhibitor of CXCR4. Treatment with plerixafor transiently decreases plasma levels of free VEGF (unbound to bevacizumab) and proangiogenic markers (angiopoetin-2 and basic fibroblast growth factor).[93]

Hypoxia hijacks immune tolerance by directly suppressing immune effector cells or augmenting immune inhibitory cells; this issue cannot be effectively addressed by radiotherapy or TMZ.[94] Based on these findings, the combination of anti-angiogenic therapy and immunotherapy (especially ICIs) may be a valid therapeutic option for enhancing cancer immunity. Treatment with angiogenic agents remodels the immune system by altering populations of immune cells and multiple cytokines.[95] VEGF-induced inhibitory checkpoints (eg, PD1 and T-cell immunoglobulin and mucin domain 3) on DCs or CD8+ T cells are found to revert once the VEGF-VEGFR interaction is blocked.[96] Under antiangiogenic therapy, normalized tumor blood vessels demonstrate restored ability of drug penetration; drug uptake by tumor cells is also improved, resolving the issue of effectiveness of chemotherapy drugs. In the ReACT study (NCT01123291), patients received bevacizumab with rindopepimut, an injectable peptide vaccine targeting EGFRvII; robust anti-EGFRvIII titers (≥1:12,800) were achieved and survival was prolonged compared to that of rindopepimut-naive patients.[97] Tumor vascular normalization can also improve tumor tissue perfusion and immune cell infiltration, thereby enhancing the effectiveness of immunotherapy; activated or reprogrammed immune cells can also normalize tumor blood vessels. In a study, patients with high CD8+ T-cell infiltration experienced significant benefit from combination therapy with bevacizumab and lomustine.[98] In another study, triple blockade of VEGF, angiopoetin-2, and PD-1 significantly extended survival duration in orthotopic GBM models compared with vascular targeting alone. In the GBM microenvironment, triple therapy induced an increase in CTL counts and decreased myeloid-derived suppressor cells and Tregs; it also offered higher global vascular normalization.[99]

However, results from other clinical trials are less satisfactory. In a study, patients who received bevacizumab plus PD1 blockade for glioma did not demonstrate a better prognosis compared to the bevacizumab-naive group; in addition, some patients discontinued study treatment due to adverse effects.[100] As shown in the GliAvAx clinical trial (NCT03291314), the combination of avelumab (anti-PD-L1 IgG1 antibody) plus axitinib (VEGFR 1-3 inhibitor) is well tolerated; however, no obvious synergistic efficacy was observed in patients with GBM.[101] The immunosuppressive character, toxic side effects, and multiple patterns of angiogenesis considerably limit the efficacy of combined approaches in GBM.

Immunotherapy + TTFields

In 2011, the FDA approved a device for the treatment of patients with GBM, namely, TTFields.[3] It delivers intermediate frequency (200 kHz in humans) and low intensity (1–3 V/cm) electric fields to the human body through transducer arrays. Under alternating electrical fields, the uniform electrical field within dividing cells is disrupted; this breaks the alignment of tubulin subunits and hinders normal microtubule spindle formation. These disruptive events preferentially occur in rapidly growing tumor cells than in normal cells, and disintegrate their daughter cells.[102] In addition to the direct anti-mitotic effect, the anti-tumor effect of TTFields may be mediated by diverse mechanisms. It may also transiently disrupt the structure of the BBB, increase membrane permeability in GBM cells, and elevate cellular concentrations of anti-tumor drugs.[103,104] In the NCT00916409 study, the addition of TTFields significantly improved progression free survival and OS compared to TMZ or radiotherapy alone.[105] TTFields-related toxicities and adverse events are milder than those of chemotherapy; this corresponded with a better quality of life in the NCT00379470 study.[106] Safety concerns have been adequately addressed based on a safety surveillance analysis among >11,000 patients with GBM who underwent TTFields-based treatment.[107]

TTFields-mediated cell death and immunogenic pyroptotic cell death can activate robust innate immunity pathways, priming effective responses to immunotherapy (Fig. 3). TTFields-treated cancer cells release more damage-associated molecule patterns such as high mobility group box protein 1 and adenosine triphosphate and demonstrate calreticulin exposure; this promotes DC maturation and engulfment of tumor cells.[108] In a study, positive T cell responses were observed in TTFields-treated tumor areas with abundant CD45+ T cells; however, CD45+ T cells had spread discretely in tumors of controls.[109] Notably, TTFields have the ability to trigger an adaptive anticancer immune response targeting residual cancer cells. CTLs isolated from TTFields-treated tumors demonstrate increased production of interferon-γ and enhanced anti-tumoral T cell function; this supports the use of combined TTFields and T-cell based immunotherapeutic approaches.[110,111] Chen et al[112,113] showed that the use of TTFields could upregulate expression levels of proinflammatory cytokines, thereby increasing the immune infiltration of activated DCs, macrophages, and T cells and turning the “cold” GBM “hot.” Changes in the quantity and activity of CD8+ T cells in the TTFields-stimulated tumors suggest a significant shift from a pro-tumoral to anti-tumoral immune signature. This indicates that the integration of TTFields with ICIs may provide superior efficacy in patients with glioma.

F3
Figure 3.:
Mechanism of anti-tumor activity and immune activation by TTFields. Through delivering intermediate frequency (200 kHz in humans) and low intensity (1–3 V/cm) electric field, TTFields disrupt proliferation of glioma cells directly. Meanwhile, cell debris is immunogenic to promote immune activity. The structure of the BBB can also be transiently broken to increase infiltration of immune cells. Created with BioRender.com. ATP=adenosine triphosphate, BBB=blood-brain barrier, GBM=glioblastoma multiforme, HMGB1=high mobility group box 1, IL-1β=interleukin 1 beta, IL-6=interleukin 6, MHC=major histocompatibility complex, NO=nitric oxide, ROS=reactive oxygen species, TNF-α=tumor necrosis factor α, TTFields=tumor treating fields.

Limitations

This review described immunotherapy in glioma and summarized clinical advances. Different types of immunotherapies have been tested against glioma, whereas current results are less satisfactory. Identifying new therapeutic targets and investigating combined therapeutic strategies are valuable for further exploration, which are also summarized in this review. However, there is still some limitation due to insufficient reports and incomplete retrieval of updating research.

Conclusions

Growing evidence indicates the feasibility of immunotherapy in patients with glioma. Based on growing recognition of the special immune landscape of glioma, the dismal response rates to traditional therapy may be surpassed by multipronged immunotherapy and innovative immunotherapeutic strategies (Fig. 4). However, existence of BBB and inadequate immune cells result in unique immune-suppression in glioma and restricting infiltration of immune cells. High heterogeneity and invasiveness of glioma result in off-target of adoptive immune cells, which also contributes to limited response to routine immune therapy. Further investigations regarding safe and effective targets and synthetic methods are warranted for developing future immunotherapeutic strategies.

F4
Figure 4.:
Graphic summary of current therapeutic options in glioma. Since last century, different strategies were innovated including chemotherapy, radiotherapy, immune therapy and tumor treating fields. As illustrated, current immunotherapy in glioma are comprised of immune regulator, chimeric antigen receptor T/NK cell, immune checkpoint inhibitor, oncolytic virus, and tumor vaccine. Created with BioRender.com. CIK-DC=cytokine-induced killer-dendritic cells, CTLA-4=cytotoxic T lymphocyte-associated antigen 4, EGFRvIII=epidermal growth factor receptor variant III, EphA2=ephrin type-A receptor 2, GD2=disialoganglioside 2, HER2=human epidermal growth factor receptor 2, IFN-γ=interferon gamma, IL13Rα2=interleukin 13 receptor α2, PD-1=programmed cell death 1, TAA=tumor associated antigen, TNF-α=tumor necrosis factor α, VEGFα=vascular endothelial growth factor α.

Acknowledgments

None.

Author contributions

YS, MW, YL, LC, XB, and CX participated in the writing of the article. XB and CX reviewed and modified the article. All authors read and approved the final article for publication.

Financial support

This work was supported by the National Natural Science Foundation of China (No. 81873048, to CX), Medico-Engineering Cooperation Funds from University of Electronic Science and Technology of China (No. ZYGX2021YGCX004, to CX), Sichuan Science and Technology Program (No. 2021YFH0187, to YS), Medico-Engineering Cooperation Funds from University of Electronic Science and Technology of China (No. ZYGX2021YGCX018, to YS), and the Fundamental Research Funds for the Central Universities (No. ZYGX2020KYQD002, to YS).

Conflicts of interest

The authors declare that there are no conflicts of interest.

Editor note: XB is an Editorial Board member of Journal of Bio-X Research. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal’s standard procedures, with peer review handled independently of this Editorial Board member and their research groups.

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Keywords:

chimeric antigen receptor T cells; glioma; immune checkpoint inhibitor; immunotherapy; neoantigen

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