Felix Corr1,2*, Philipp Einheuser1, Beat Alessandri1, Florian Ringel1 and Harald Krenzlin1
Received:May 17, 2021 Published:May 21, 2021
Corresponding author:Felix Corr, Department of Neurosurgery, Johannes Gutenberg-University, Langenbeckstrase 1, 55131 Mainz, Germany
Glioblastoma is the most common malignant brain tumor associated with poor prognosis and mortality. Despite optimal primary therapy, median survival remains poor. New approaches to tumor eradication are lacking and urgently needed to improve patient survival and quality of life. Immunotherapies promise new treatment strategies in addition to standard treatment. Checkpoint inhibition and programmed death ligand 1 blockage so far have failed to mount a clinical impact in glioblastoma. Modified T-cells are seen as a novel promising therapy as they have not been extensively tested in glioblastoma. Today, such modified T-cells are established in the treatment hematological malignancies. Approaches include adoptive transfer of T-cells genetically engineered with a receptor that targets tumor-associated antigens (TAAs).
Chimeric antigen receptor generated by transferring domains derived from antibodies and T-cell receptors that interact with specific TAAs can provide specific recognition of TAAs. To overcome obstacles in terms of specificity of the TCRs, single chain T-cell receptors might be a promising approach. The role of cytomegalovirus (CMV) for immunotherapy is debated in the literature. Establishment of CMV as a potential antigen for targeted immunotherapy in glioblastoma could be a promising approach in combination with CAR T-cell therapy. In this context, single chain fragment variable may play an important role in the recognition of target antigens. We sought to examine the current state of the literature with respect to CAR T-cell therapy for glioblastoma associated with a CMV positive serotype. Clinically, such an association could lead to the production of new experimental therapies.
Keywords: Glioblastoma; GBM; CAR T-cell; Chimeric Antigen Receptor; T-lymphocytes; Cytomegalovirus; CMV; Immunotherapy; Targeted Therapy; Central Nervous System Neoplasms
Among the group of brain-derived tumors, glioblastoma stands out as the most malignant tumor among astrocytoma in terms of both progression free survival, overall survival, mortality and is associated with high economic costs [1-2]. Complexity of neurooncological treatment is evident from the multimodal initial therapy, although the course of therapy is individualized, especially with regard to radio- and chemotherapy, due to histopathological characteristics of tumor biology such as MGMT promoter status [3,4]. Moreover, intratumorally heterogeneity , blood brain barrier  and immune evasion pose particular challenges for therapeutic approaches . High morbidity and mortality of the tumor therefore requires further experimental approaches, which can be considered as additional therapy options besides the actual standard treatment. The role of immunotherapy is a current point of discussion due to the observed immunosuppressive role of glioblastoma . Immunotherapies including checkpoint inhibition and programmed death ligand 1 (PD-L1) blockage so far have failed to mount a clinical impact in glioblastoma .
Experimental studies showed the release of anti-inflammatory cytokines, reducing levels of antigen-presenting molecules, and engaging immune checkpoint–activating regulatory receptors on T-cells via PD-L1. Glioblastoma derived extracellular vesicle (EVs) carrying PD-L1 are able to prevent T-cell activation mediating antitumor immunity suppression . While T-cells are present in glioblastoma, programmed cell death protein 1 (PD-1) blockade did not lead to sufficient T-cell activation and failed to demonstrate anti-tumoral efficacy in phase III clinical trials . T-cell activation and anti-tumor response represents the goal of targeted immunotherapy. Short dendritic cells (DCs) present captured tumor associated antigens to prime and subsequently activate T-cells. Tumor infiltration of cytolytic T-cells with TCR recognition of tumor antigens leads to tumor destruction with a subsequent release of more TAAs for DCs to present . The adoptive transfer of genetically engineered T-cells with a receptor targeting a specific TAA bypassing the education of antigen specific T-cells by DCs establishes another valuable concept for immunotherapeutic approaches. These T-cells use a chimeric antigen receptor which is generated by transferring domains derived from antibodies and T-cell receptors known to interacting with specific TAA [11-13].
Currently, modified T-cells are established in the treatment of acute lymphoblastic leukemia , chronic lymphocytic leukemia , B-cell lymphoma  and multiple myeloma . However, problems in the engineering of CAR T-cells have to be considered. First, identification of a suitable tumor antigen is not distinct. Mispairing of exogenous TCR chains with endogenous TCR chains could lead to a graft-versus-host reaction . To avoid this, single chain T-cell receptor could lead to the elimination of heterodimers, increasing the specificity of targeting specific antibodies [19, 20]. The interface of the immunosuppressive role of glioblastoma and possible immunotherapy with CAR T-cells is potentially filled by the presence of the cytomegalovirus. Human herpesvirus 5 (CMV, cytomegalovirus) remains after infection in the human body throughout life, so that periodic reactivations may occur due to stress and immunocompromise .
Prevalence of CMV infection in the general population is estimated between 45% and 100% . Of interest, proteins and nucleic acids of human cytomegalovirus have been detected in several tumor types, including colon cancer , prostate cancer  and glioblastoma [25, 26]. While proteins were found in the tumor area, they are undetectable outside the tumor in the brain . With respect to an immunosuppressed microenvironment, reactivation of CMV from the latent phase seems likely to occur. Thus, CMV infection is central to tumor biology processes in terms of cell proliferation, invasion and angiogenesis . Various clinical data already propagate the clinical significance for the possible approach of immunotherapy in relation to cytomegalovirus. Thus, CMV could be established as a possible antigen for targeted immune-therapy in glioblastoma using CAR T-cells.
Targeted Immunotherapy using Chimeric Antigen Receptor T-Cells
CAR T-cells reveal a promising approach in immunotherapy. To date, there are only a few clinically approved applications of CAR T-cells. In the treatment of B-cell precursor acute lymphoblastic leukemia, tisagenlecleucal, a CD19-targeted CAR T cell therapy, has been approved . Clinical evidence is very limited in glioblastoma, with mainly experimental in vitro and in vivo studies. The main hurdles in T-cell therapy in glioblastoma are considered to be tumor heterogeneity, blood-brain barrier, immunosuppressive microenvironment and antigen escape . As a new target, fibroblast activation protein (FAP) was identified as a new target for CAR T-cells, which appeared to be overexpressed in the tumor area . Potent anti-GBM activity with chlorotoxin-directed CAR T-cells was recently demonstrated, particularly in tumors lacking expression of other GBM-associated antigens . In the murine model, an improved response to therapy with CAR T-cells targeting EGFRvIII with the aid of interleukin 12 (IL-12) was recognized. IL-12 not only increased the cytotoxicity of CAR T-cells but also reformed the immunocompromised microenvironment .
To increase the efficacy of CAR T-cells, the inhibition of PP2A with LB-100 was investigated, showing that LB-100 improved anti CAIX CAR T-cell treatment both in vitro and in vivo . PD-1 knockout seems to increase the lytic activity of EGFRvIII-CAR T-cells against PD-L1+ EGFRvIII+ GBM cells . In the context of PD-1 checkpoint blockade, CAR T-cells with PD-1 blockade not only showed higher killing efficiency in vitro, but also resulted in an increased immunological response by tumor infiltrating lymphocytes (TILs) in the mouse model . The problem of the immunosuppressive environment of GBM tumors was addressed by Li et al. in their study on TGFβ-resistant CAR-Ts for GBM therapy. They showed enhanced anti-tumor efficacy of EGFRvIII-specific CAR-T and prolonged survival in mice bearing GBM. Furthermore, a polarizing pro-inflammatory and anti-tumorigenic response was evident by an increased expression of M1 polarization markers . A complication of CAR-T cell therapy is allorejection in the case of a graft-versus-host reaction.
Insufficient specificity of the T-cells from the receptor to the antibodies causes the response also to non-tumoral antibodies, so that a cytokine-release syndrome (CRS) can occur, a systemic inflammatory reaction due to an immune cell activation . Therefore, high specificity must be ensured in the engineering of CAR T-cells. Choe et al. reported controlled tumor cell killing by SynNotch CAR T-cells. In the mouse model, a single intravenous infusion of EGFRvIII synNotch CAR T-cells showed higher antitumor efficacy and T-cell durability than in conventionally expressed CAR T-cells without off-tumor killing . In vitro studies confirm that CMV pp65 specific T-cells are able to kill glioblastoma cells. However, clinical studies further confirming this are lacking. A recent case report demonstrated the response of antitumor therapy of allogenic natural killer cells and pp65 pulsed dendritic cells. After therapy, no residual tumor was seen on imaging  (Figure 1).
Figure 1: The cancer immunity cycle. Immunotherapy is used to directly target tumor cells in an individualized manner while concomitantly strengthening the host anti-tumor response and thus developing synergistic effects.
The role of Cytomegalovirus
The role of detection of components of CMV in glioblastoma comes into question as a link to immunotherapy. Krenzlin et al. recently demonstrated in vivo the significant increase of tumor volume with a worse overall survival in CMV infected mice. PDGF as CMV induced factor seemed to play a major role in pericyte recruitment, angiogenesis and tumor growth . A prospective randomized study showed the outcome after vaccination with CMV pp65 RNA loaded dendritic cells. Significant increases in IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T-cells were observed. These results could serve as potential biomarkers for the effective response of immunotherapy . Long-term survival in GBM patients after CMV pp65-targeted vaccination was studied when in addition to temozolomide, 11 patients received three doses of the vaccine, showing an increased ratio and proliferation of regulatory T-cells. The patients showed long-term progression free survival and overall survival .
Figure 2: CMV detection in murine GBM cells. GL261-Luc2 murine glioblastoma cells 24 hours (a) and 48 hours (b) after mCMV-eGFP infection. MOI=1. (Einheuser et al, unpublished).
Autologous polyclonal CMV pp65-specif T-cells were studied after administration after manufacture, three weeks of lymphodepletion, and dose-tense temozolomide in a Phase I/II trial. Increased circulating numbers of CMV+ CD8+ T-cells were seen but suppressed effector activity was evident. Furthermore, CMV seropositivity did not guarantee a tumor response to CMVspecific T-cells . In a prospective study with autologous CMVspecific T-cells, twenty-eight patients received adjuvant treatment. The long-term survival of patients treated before tumor recurrence was significantly increased . With regard to the tumor immune microenvironment, it could be shown that a lower number of tumorinfiltrating CD3+ T-cells in recurrent tumors was evident in patients with increased overall survival after T-cell therapy. In addition, these patients showed low to undetectable PD-L1 expression in tumor cells, suggesting that absence of PD-L1 immunosuppression favors effective immune control with adoptive T-cells .
Benefits of using single chain T-cell receptors
To reduce or eliminate the probability of graft-versus-host reactions, the specificity of the TCR has to be adjusted. Aggen et al. describe the possibility of a single-chain VαVβ T cell receptor. By combining stabilized Vα/Vβ single-chain TCR (scTv) with intracellular signaling domains such as Lck and CD28, mispairing of exogenous TCR chains with endogenous ones could be prevented, so that a functional T cell response could be activated (19). Additionally, Knies et al. compared gp100(280-288) with p53(264- 272) tumor antigen-specific scTCR with regard to mispairing with TCRα. This showed that mispairing was largely reduced after transduction into human TCRα/β-positive T-cells. The generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to enhance T-cell activation in CMV-infected tumor patients. The authors conclude that optimized scTCR/Cα inhibits residual TCR mismatching to achieve safe adoptive immunotherapy for large endogenous TCRα/β-positive T-cells .
New approaches in immunotherapy for glioblastoma are lacking. Weaknesses and obstacles need to be overcome in therapy with CAR-T-cells. In this context, single chain TCRs may play an important role. Regarding tumor immunocompromised microenvironment, cytomegalovirus appears to play an essential role in oncomodulation in glioblastoma. This knowledge can be implemented in experimental research to act as a target for potential immunotherapy with CAR T-cells.
PD-1: Programmed death ligand 1
TAA : Tumor associated antigens
CAR: Chimeric antigen receptor
TCR: T-cell receptor
scFv: Single chain fragment variable
PD-L1: Programmed cell death ligand 1
DCs: Dendritic cells
EVs: Extracellular vesicles
FAP: Fibroblast activation protein
IL-12: Interleukin 12
PP2A: Protein phosphatase 2
EGFRvIII: Epidermal growth factor receptor variant III
TILs: Tumor infiltrating lymphocytes
CRS: Cytokine-release syndrome
TNFα: Tumor necrosis factor α
CCL3: C-C Motif Chemokine Ligand 3
CD8: Cluster of differentiation 8
CD3: Cluster of differentiation 3
Lck: Lymphocyte-specific protein tyrosine kinase
CD28: Cluster of differentiation 28
CAIX: Carbonic anhydrase IX
TGFβ: Transforming Growth Factor β
PDGF: Platelet-derived growth factor
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