Harnessing Radiation Biology to Augment Immunotherapy for Glioblastoma.

Glioblastoma is the most common adult primary brain tumor and carries a dismal prognosis. Radiation is a standard first-line therapy, typically deployed following maximal safe surgical debulking, when possible, in combination with cytotoxic chemotherapy. For other systemic cancers, standard of care is being transformed by immunotherapies, including checkpoint-blocking antibodies targeting CTLA-4 and PD-1/PD-L1, with potential for long-term remission. Ongoing studies are evaluating the role of immunotherapies for GBM. Despite dramatic responses in some cases, randomized trials to date have not met primary outcomes. Challenges have been attributed in part to the immunologically "cold" nature of glioblastoma relative to other malignancies successfully treated with immunotherapy. Radiation may serve as a mechanism to improve tumor immunogenicity. In this review, we critically evaluate current evidence regarding radiation as a synergistic facilitator of immunotherapies through modulation of both the innate and adaptive immune milieu. Although current preclinical data encourage efforts to harness synergistic biology between radiation and immunotherapy, several practical and scientific challenges remain. Moreover, insights from radiation biology may unveil additional novel opportunities to help mobilize immunity against GBM.

Inhibitory Receptors Induced by VSV Viroimmunotherapy Are Not Necessarily Targets for Improving Treatment Efficacy.

Systemic viroimmunotherapy activates endogenous innate and adaptive immune responses against both viral and tumor antigens. We have shown that therapy with vesicular stomatitis virus (VSV) engineered to express a tumor-associated antigen activates antigen-specific adoptively transferred T cells (adoptive cell therapy, ACT) in vivo to generate effective therapy. The overall goal of this study was to phenotypically characterize the immune response to VSV+ACT therapy and use the information gained to rationally improve combination therapy. We observed rapid expansion of blood CD8+ effector cells acutely following VSV therapy with markedly high expression of the immune checkpoint molecules PD-1 and TIM-3. Using these data, we tested a treatment schedule incorporating mAb immune checkpoint inhibitors with VSV+ACT treatment. Unlike clinical scenarios, we delivered therapy at early time points following tumor establishment and treatment. Our goal was to potentiate the immune response generated by VSV therapy to achieve durable control of metastatic disease. Despite the high frequency of endogenous PD-1+ TIM-3+ CD8+ T cells following virus administration, antibody blockade did not improve survival. These findings provide highly significant information about response kinetics to viroimmunotherapy and juxtapose the clinical use of checkpoint inhibitors against chronically dysfunctional T cells and the acute T cell response to oncolytic viruses.

Immunogenicity of self tumor associated proteins is enhanced through protein truncation.

We showed previously that therapy with Vesicular Stomatitis Virus (VSV) expressing tumor-associated proteins eradicates established tumors. We show here that when cellular cDNA were cloned into VSV which retained their own poly-A signal, viral species emerged in culture which had deleted the cellular poly-A signal and also contained a truncated form of the protein coding sequence. Typically, the truncation occurred such that a Tyrosine-encoding codon was converted into a STOP codon. We believe that the truncation of tumor-associated proteins expressed from VSV in this way occurred to preserve the ability of the virus to replicate efficiently. Truncated cDNA expressed from VSV were significantly more effective than full length cDNA in treating established tumors. Moreover, tumor therapy with truncated cDNA was completely abolished by depletion of CD4+ T cells, whereas therapy with full length cDNA was CD8+ T cell dependent. These data show that the type/potency of antitumor immune responses against self-tumor-associated proteins can be manipulated in vivo through the nature of the self protein (full length or truncated). Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-tumor-associated proteins can be broken through manipulation of protein integrity, allowing for rational design of better self-immunogens for cancer immunotherapy.

Harnessing the Power of Onco-Immunotherapy with Checkpoint Inhibitors.

Oncolytic viruses represent a diverse class of replication competent viruses that curtail tumor growth. These viruses, through their natural ability or through genetic modifications, can selectively replicate within tumor cells and induce cell death while leaving normal cells intact. Apart from the direct oncolytic activity, these viruses mediate tumor cell death via the induction of innate and adaptive immune responses. The field of oncolytic viruses has seen substantial advancement with the progression of numerous oncolytic viruses in various phases of clinical trials. Tumors employ a plethora of mechanisms to establish growth and subsequently metastasize. These include evasion of immune surveillance by inducing up-regulation of checkpoint proteins which function to abrogate T cell effector functions. Currently, antibodies blocking checkpoint proteins such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) and anti-programmed cell death-1 (PD-1) have been approved to treat cancer and shown to impart durable clinical responses. These antibodies typically need pre-existing active immune tumor microenvironment to establish durable clinical outcomes and not every patient responds to these therapies. This review provides an overview of published pre-clinical studies demonstrating superior therapeutic efficacy of combining oncolytic viruses with checkpoint blockade compared to monotherapies. These studies provide compelling evidence that oncolytic therapy can be potentiated by coupling it with checkpoint therapies.

Preservation of dendritic cell function during vesicular stomatitis virus infection reflects both intrinsic and acquired mechanisms of resistance to suppression of host gene expression by viral M protein.

Inhibition of host-directed gene expression by the matrix (M) protein of vesicular stomatitis virus (VSV) effectively blocks host antiviral responses, promotes virus replication, and disables the host cell. However, dendritic cells (DC) have the capacity to resist these effects and remain functional during VSV infection. Here, the mechanisms of DC resistance to M protein and their subsequent maturation were addressed. Flt3L-derived murine bone marrow dendritic cells (FDC), which phenotypically resemble resident splenic DC, continued to synthesize cellular proteins and matured during single-cycle (high-multiplicity) and multicycle (low-multiplicity) infection with VSV. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived myeloid DC (GDC), which are susceptible to M protein effects, were nevertheless capable of maturing, but the response was delayed and occurred only during multicycle infection. FDC resistance was manifested early and was type I interferon (IFN) receptor (IFNAR) and MyD88 independent, but sustained resistance required IFNAR. MyD88-dependent signaling contributed to FDC maturation during single-cycle infection but was dispensable during multicycle infection. Similar to FDC, splenic DC were capable of maturing in vivo during the first 24 h of infection with VSV, and neither Toll-like receptor 7 (TLR7) nor MyD88 was required. We conclude that FDC resistance to M protein is controlled by an intrinsic, MyD88-independent mechanism that operates early in infection and is augmented later in infection by type I IFN. In contrast, while GDC are not intrinsically resistant, they can acquire resistance during multicycle infection. In vivo, splenic DC resist the inhibitory effects of VSV, and as in multicycle FDC infection, MyD88-independent signaling events control their maturation.

Complexes of vesicular stomatitis virus matrix protein with host Rae1 and Nup98 involved in inhibition of host transcription.

Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.