Vile, Richard G.

Despite decades of research and numerous clinical trials, the prognosis of patients diagnosed with glioblastoma (GBM) remains dire with median observed survival at 8 months. There is a critical need for novel treatments for GBM, which is the most common malignant primary brain tumor. Major advances in cancer therapeutics such as immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy have not yet led to improved outcomes for GBM. Conventional therapy of surgery followed by chemoradiation with or without tumor treating fields remains the standard of care. One of the many approaches to GBM therapy currently being explored is viral therapies. These typically work by selectively lysing target neoplastic cells, called oncolysis, or by the targeted delivery of a therapeutic transgene via a viral vector. In this review, we discuss the underlying mechanisms of action and describe both recent and current human clinical trials using these viruses with an emphasis on promising viral therapeutics that may ultimately break the field’s current stagnant paradigm.

Abstract Oncologic viruses (OV) can induce tumor death through both direct oncolysis and immune-mediated destruction. Our study investigates the use of a novel OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-b) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNb-TYRP1). VSV is an attractive vector since humans are not naturally infected with VSV; thus, pre-existing immunity is minimal. IFN-b leads to increased T-cell responses and tumor cell apoptosis, while TYRP1, a melanocyte differentiation antigen, increases immunogenicity of the therapy. We conducted a Phase 1 clinical trial with a 3+3 design in patients with metastatic uveal melanoma. VSV-IFNb-TYRP1 was injected into a liver metastasis under image guidance, then administered on the same day as a single intravenous (IV) infusion. IV doses started at dose level (DL) 1 1 × 1010 TCID50, then escalated to DL2 3 × 10 10, DL3 1 × 1011, and DL4 3 × 1011. The dose delivered intratumorally (IT) varied based on the IV dose. The primary endpoints were safety and maximum tolerated dose (MTD). Efficacy was a secondary endpoint. Correlative studies focused on understanding viral pharmacokinetics (PK) and immunological responses induced by VSV-IFNb-TYRP1 therapy. Twelve patients with previously treated metastatic uveal melanoma were enrolled. Median follow up was 19.1 months. Four DLs were evaluated with one patient at DL4 experiencing dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). Non-DLTs at DL4 were fatigue, fever, CRS, hematological toxicities (decreased platelets and lymphocytes), and AST elevation. Four patients had stable disease (SD) and 8 had progressive disease (PD) as their best response. ELIspot data show that three of the four patients with response to TYRP1 also had a response to gp100, suggesting possible epitope spreading. These four patients received immune checkpoint inhibitor treatment as the next line of therapy, which led to clinical benefit for two patients. Our study evaluated VSV-IFNb-TYRP1 administered via IT and IV routes in a previously treated population of metastatic uveal melanoma patients and found no major safety events. Although there were no clear objective responses to VSV-IFNb-TYRP1, dose-dependent immunogenicity to melanoma antigens was seen. While OV response alone is not sufficient for clinical benefit, the evidence of epitope spreading is encouraging. Future studies of VSV-IFNb-TYRP1 will evaluate combinations with other therapies. Citation Format: Katherine E. Smith, Adam Weisbrod, Carrie Strand, Jacob Allerd, Jose S. Pulido, Alysha Newsom, Lianwen Zhang, Nandakumar Packiriswamy, Heather Montane, Lisa Kottschade, Anastasios Dimou, Yiyi Yan, Markovic Svetomir, Kah Whye Peng, Richard G. Vile, Matthew S. Block, Roxana S. Dronca. Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT207.

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti—programmed cell death protein 1 (anti–PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti–PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I–restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I–independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Abstract We have previously shown that vesicular stomatitis virus (VSV) vectors encoding truncated tumor antigens, NRAS, Cytochrome C1, and TYRP1, prime superior systemic anti-tumor T cell responses and provide enhanced therapeutic benefit compared to vectors expressing full-length forms of the protein. Mapping of the roughly 20 amino C-terminal truncated regions onto structural domains of each protein suggested the truncations disrupted the stability and localization of each protein. Indeed, immunofluorescent staining and radiolabeled pulse chase found that truncated TYRP1 (ΔTYRP1) had an altered localization pattern and a significantly reduced half-life compared to full-length TYRP1. Thus, we hypothesized that expression of destabilized antigens from VSV would provide superior therapy compared to expression of wild-type antigens. To test this, we introduced point-mutations into structurally important domains of the model antigen ovalbumin (OVA). We identified one mutant which was significantly destabilized (OVAmut), which resulted in enhanced MHC Class I presentation of the immunogenic SIINFEKL epitope on transfected cell in vitro. Expression of OVAmut resulted in better recognition by cognate CD8 OTI T cells, enhanced T cell activation, proliferation, and effector cytokine production. Finally, a VSV vector expressing OVmut was better able to prime OTI T cells in vitro as well as in vivo. Ongoing studies aim to identify the changes in the quantity and repertoire of immunogenic epitopes presented from cells infected with VSV vectors expressing destabilized antigens. These studies provide insight into the capacity of the VSV platform to successfully encode unstable antigens with the goal improve immunogenic responses.

Abstract Immunosuppression following damage to the CNS is a common, yet poorly understood, feature of neurological diseases as diverse as stroke, traumatic brain injury, and glioblastoma. This immunosuppression is a barrier to successful patient outcomes. We sought to define the effect of various brain insults on the thymus and T-cell responses. We tested thymic function following various neurological insults, including viral infection, brain tumor, sterile inflammation, physical injury, and seizures. All brain insults resulted in significant thymic involution that was reversible if the insult was cleared. Thymic involution did not occur following similar peripheral insults. Using parabiosis, we demonstrated that thymic involution was transferable via circulatory routes from glioma-bearing to non-tumor-bearing parabionts. Similarly, serum obtained from mice with ongoing neurological insults potently inhibited T-cell proliferation in vitro. We next fractionated the serum based on molecular weight and tested the resulting fractions’ immunosuppressive potential. Interestingly, we found that serum fractions with large molecular weights of greater than 100 kiloDaltons were responsible for the immunosuppressive properties of serum obtained from glioma-bearing mice. In short, CNS-specific insults, regardless of nature, induce immunosuppression by prompting thymic involution and systemic immunosuppression mediated through circulating factors with large molecular weight. These studies provide evidence of the mechanisms leading to immune deficiencies observed in patients following neurological injuries.

Abstract T cells expressing chimeric antigen receptors (CAR T cells) are manufactured outside the body using non-physiological activation and in vitro culture protocols. The procedure takes 3 weeks and costs approximately $450,000 to administer. We have devised a scheme using mouse models for manufacturing CAR T cells in just 3 days employing a natural, polyclonal anti-viral immune response inside a lymph node (LN) using off the shelf reagents that can be applied widely in populations. CAR T cells emerge from the LN within a week, invade solid tumor, and eliminate the targeted antigen-positive tumor cells. Polyclonal activation of T cells is achieved by modifying the antigen presenting properties of lymph node resident professional antigen presenting cells (APC) through viral expression of allogeneic major histocompatibility antigens. The ensuing polyclonal activation occurs in the context of virus activated APC within the normal aarchitecture of the LN and confers robust T cell activation signals directing the development of cytolytic T cells. During the course of T cell activation, the T cells are transduced in situ with retroviruses encoding selected CARs. This approach could be applied to any number of different CARs to target a variety of tumors. T cells activated in the lymph node are functionally distinct from those activated using standard CAR T manufacturing protocols. We anticipate that CAR T cells generated in situ to natural stimuli will exhibit more normal immune regulatory properties that will impact their therapeutic utility. Because our approach uses off the shelf reagents and is administered using standard clinical procedures, cost to patients should be lowered and access to therapy increased.

Abstract Glioblastoma is the most common and lethal primary brain tumor in adults, with a median survival less than 15 months despite current standard of care. Oncolytic immunovirotherapies (OVs) have shown promise in early clinical trials. OVs are engineered to specifically target cancer cells, trigger autophagic programmed cell death, and release tumor antigens in the context of T-cell activating cytokines. Cellular communication network factor 1 (CCN1) is an extracellular matrix protein expressed in 70% of glioblastomas that has been shown to drastically reduce OV efficacy, particularly herpes simplex virus type 1 (HSV-1). Our objectives were to decode protein-protein interaction (PPI) networks activated by CCN1, identify critical nodes in resistance networks, and use this analysis to design OVs that overcome CCN1-mediated resistance. We used NetDecoder to elucidate phenotype-specific PPI subnetworks in LN229 human glioblastoma cells with tetracycline-inducible CCN1 expression. Publicly available microarray data on CCN1-induced and control samples established differentially expressed genes, which served as sources in our PPI network to derive prioritized context-specific subnetworks. We found that 11 source genes collaborate via 39 deep nodes to confer OV resistance to CCN1-expressing glioblastomas. Of these, a router (IKBKE) and a sink (YBX1) have been previously implicated in glioblastoma pathogenesis. We conclude that autophagy regulator HSP90AA1, a critical node, may be targeted to improve OV efficacy in CCN1-expressing glioblastomas. Furthermore, WebGestalt overrepresentation analysis of subnetwork nodes suggests that oncolytic adenovirus may be more effective than HSV-1 as a synthetic biology chassis.

Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brainstem tumor that is the leading cause of death among pediatric brain malignancies. Despite over 200 clinical trials, no significant progress has been made, leading our lab to consider suicide gene therapy. The brainstem has particularly sensitive anatomy, creating the potential for toxic inflammation. Furthermore, the clinical standard of care for DIPG includes conventional radiation therapy (RT) followed by dexamethasone (DEX), each of which can alter the efficacy of gene therapy. In this study we found that a variety of gliomas, including human DIPG and murine glioma cell lines, are infectable in vitro with a replication-competent Moloney murine leukemia virus (MoMLV) (currently in Phase 2/3 clinical trials) and susceptible to ganciclovir (GCV)-mediated killing via the HSV thymidine kinase (tk) suicide gene. A B16 melanoma cell line expressing HSV tk implanted into the brainstem of mice, extended median survival with GCV therapy from 13 days to 38.5 days (p=.0006) with no signs of toxic inflammation. Pre-treatment with RT and DEX did not affect therapeutic efficacy. We have shown that GCV/TK therapy is mediated by NK and CD8 cells in subcutaneous tumors, and we are investigating whether a similar immunologic mechanism is involved in this brainstem model. These preclinical data will pave the way for novel clinical trials for the treatment of DIPG.

Abstract We study mechanisms of tumor escape from adoptive therapy in the mouse melanoma model B16ova. In this model, transfer of OT-I T cells can significantly prolong the survival of mice. However, intense OT-I pressure promotes tumor cell genomic instability, and most mice eventually develop recurrent ova gene-loss tumors. We hypothesized that simultaneous transfer of two CTL clones would limit tumor recurrence by preventing the selection of single gene-loss cells. To accomplish this, we co-transferred OT-I T cells and Pmel T cells. Co-transfer three days following tumor challenge prevented tumor growth, while neither clone alone could. And in mice with established tumors, co-transfer provided significantly better therapy than either clone alone. This improvement was due to both fewer tumors recurring and the delayed appearance of those that did recur. Additionally, we found that recurrent tumors from mice treated with co-transfer continued to express both target antigens, and the mice maintained both T cell populations. We therefore hypothesized that it should be possible to exploit those cells to further prolong the survival of mice. We used cyclophosphamide to stimulate reactivation of the immune system in mice with recurrent tumors and found that this supplemental therapy significantly prolonged the lives of only those mice which had previously received co-transfer. Overall, our results reveal several benefits of targeting multiple tumor antigens.

Abstract Previously, we reported that killing tumor cells in vivo with the HSV thymidine kinase/ganciclovir system generates potent antitumor immunity, determined in part by the mechanism by which the cells die and by the levels of inducible heat shock protein (hsp) expression induced during the process of cell death. Here, we show that induction of hsp70 expression induces an infiltrate of T cells, macrophages, and predominantly dendritic cells (DCs) into the tumors as well as an intratumoral profile of Th1 cytokine expression (IFN-γ, TNF-α, and IL-12) and enhances immunogenicity via a T cell-mediated mechanism. In addition, the protection conferred by hsp70 is both tumor and cell specific. We also demonstrate that hsp70 targets immature APC to make them significantly more able to capture Ags. This is likely to optimize cross-priming of the infiltrating APC with tumor Ags, which are simultaneously being released by the dying cells. In addition, using an Myc epitope-tagged hsp70 expression vector, we present evidence that hsp70 released from dying tumor cells is taken up directly into DCs and may, therefore, be involved in direct chaperoning of Ags into DCs. Taken together, our data suggest that hsp70 induction serves to signal the immune system of the presence of an immunologically relevant (dangerous) situation against which an immune reaction should be raised.