Combination Therapy With Reovirus and Anti-PD-1 Blockade Controls Tumor Growth Through Innate and Adaptive Immune Responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both preclinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and antitumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive antitumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (P < 0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced T(reg) activity, and increased the adaptive CD8(+) T-cell-dependent antitumor T-cell response. PD-1 blockade also enhanced the antiviral immune response but through effector mechanisms which overlapped with but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus viroimmunotherapy.

Cytokine conditioning enhances systemic delivery and therapy of an oncolytic virus.

Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells--but not from plasma--suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b(+) cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.

The profile of tumor antigens which can be targeted by immunotherapy depends upon the tumor's anatomical site.

Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here, we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens-VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1-from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α(Hi), SOX-10(Hi), c-myc(Hi), TYRP1, N-RAS(lo)Cytc(lo) antigen profile, which differed significantly from the HIF-2α(lo), SOX-10(lo), c-myc(lo), TYRP1, N-RAS(Hi)Cytc(Hi) phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b(+) cells within the local brain tumor microenvironment. Combining T-cell costimulation with systemic VSV-cDNA treatment, long-term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.

The efficacy versus toxicity profile of combination virotherapy and TLR immunotherapy highlights the danger of administering TLR agonists to oncolytic virus-treated mice.

Injection of oncolytic vesicular stomatitis virus (VSV) into established B16ova melanomas results in tumor regression, in large part by inducing innate immune reactivity against the viral infection, mediated by MyD88- and type III interferon (IFN)-, but not TLR-4-, signaling. We show here that intratumoral (IT) treatment with lipopolysaccharide (LPS), a TLR-4 agonist, significantly enhanced the local therapy induced by VSV by combining activation of different innate immune pathways. Therapy was further enhanced by co-recruiting a potent antitumor, adaptive T-cell response by using a VSV engineered to express the ovalbumin tumor-associated antigen ova, in combination with LPS. However, the combination of IT LPS with systemically delivered VSV resulted in rapid morbidity and mortality in the majority of mice. Decreasing the intravenous (IV) dose of VSV to levels at which toxicity was ameliorated did not enhance therapy compared with IT LPS alone. Toxicity of the systemic VSV + IT LPS regimen was associated with rapidly elevated levels of serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, which neither systemic VSV, nor IT LPS, alone induced. These data show that therapy associated with direct IT injections of oncolytic viruses can be significantly enhanced by combination with agonists of innate immune activation pathways, which are not themselves activated by the virus alone. Importantly, they also highlight possible, unforeseen dangers of combination therapies in which an immunotherapy, even delivered locally at the tumor site, may systemically sensitize the patient to a cytokine shock-like response triggered by IV delivery of oncolytic virus.

Functional cloning of recurrence-specific antigens identifies molecular targets to treat tumor relapse.

Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expressed cDNA library derived from recurrent tumor cells. By screening the protective, recurrence-derived VSV-cDNA library, here we identify topoisomerase-IIα (TOPO-IIα) as a recurrence-specific tumor antigen against which tolerance can be broken. Tumor recurrences, in two different types of tumor (prostate and melanoma), which had evaded two different frontline treatments (immunotherapy or chemotherapy), significantly overexpressed TOPO-IIα compared with their primary tumor counterparts, which conferred a novel sensitivity to doxorubicin (DOX) chemotherapy upon the recurrent tumors. This was exploited in vivo using combination therapies to cure mice, which would otherwise have relapsed, after suboptimal primary therapy in both models. Our data show that recurrent tumors-across histologies and primary treatments-express distinct antigens compared with the primary tumor which can be identified using the VSV-cDNA library technology. These results suggest that it may be possible to design a few common second-line therapies against a variety of tumor recurrences, in some cases using agents with no obvious activity against the primary tumor.

Phase II trial of intravenous administration of Reolysin(®) (Reovirus Serotype-3-dearing Strain) in patients with metastatic melanoma.

Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 10(10) TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.

Trap and ambush therapy using sequential primary and tumor escape-selective oncolytic viruses.

In multiple models of oncolytic virotherapy, it is common to see an early anti-tumor response followed by recurrence. We have previously shown that frontline treatment with oncolytic VSV-IFN-ß induces APOBEC proteins, promoting the selection of specific mutations that allow tumor escape. Of these mutations in B16 melanoma escape (ESC) cells, a C-T point mutation in the cold shock domain-containing E1 (CSDE1) gene was present at the highest frequency, which could be used to ambush ESC cells by vaccination with the mutant CSDE1 expressed within the virus. Here, we show that the evolution of viral ESC tumor cells harboring the escape-promoting CSDE1C-T mutation can also be exploited by a virological ambush. By sequential delivery of two oncolytic VSVs in vivo, tumors which would otherwise escape VSV-IFN-ß oncolytic virotherapy could be cured. This also facilitated the priming of anti-tumor T cell responses, which could be further exploited using immune checkpoint blockade with the CD200 activation receptor ligand (CD200AR-L) peptide. Our findings here are significant in that they offer the possibility to develop oncolytic viruses as highly specific, escape-targeting viro-immunotherapeutic agents to be used in conjunction with recurrence of tumors following multiple different types of frontline cancer therapies.

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

Adoptive T cell therapy promotes the emergence of genomically altered tumor escape variants.

Adoptive T cell therapy has been proven effective against melanoma in mice and humans. However, because most responses are incomplete or transient, cures remain rare. To maximize the efficacy of this therapy, it will be essential to gain a better understanding of the processes which result in tumor relapse. We studied these processes using B16ova murine melanoma and adoptive transfer of OT-I T cells. Transfer of T cells as a single therapy provided a significant survival benefit for mice with established subcutaneous tumors. However, tumors which initially regressed often recurred. By analyzing tumors which emerged in the presence of a potent OT-I response, we identified a novel tumor escape mechanism in which tumor cells evaded T cell pressure by undergoing major genomic changes involving loss of the gene encoding the target tumor antigen. Furthermore, we show that these in vivo processes can be recapitulated in vitro using T cell/tumor cell co-cultures. A single round of in vitro co-culture led to significant loss of the ova gene and a tumor cell population with rapidly induced and diverse karyotypic changes. Although these current studies focus on the model OVA antigen, the finding that T cells can directly promote genomic instability has important implications for the development of adoptive T cell therapies.

Tumor-targeted, systemic delivery of therapeutic viral vectors using hitchhiking on antigen-specific T cells.

Antigen-specific T cells circulate freely and accumulate specifically at sites of antigen expression. To enhance the survival and targeting of systemically delivered viral vectors, we exploited the observation that retroviral particles adhere nonspecifically, or 'hitchhike,' to the surface of T cells. Adoptive transfer of antigen-specific T cells, loaded with viruses encoding interleukin (IL)-12 or Herpes Simplex Virus thymidine kinase (HSVtk), cured established metastatic disease where adoptive T-cell transfer alone was not effective. Productive hand off correlated with local heparanase expression either from malignant tumor cells and/or as a result of T-cell activation by antigen, providing high levels of selectivity for viral transfer to metastatic tumors in vivo. Protection, concentration and targeting of viruses by adsorption to cell carriers represent a new technique for systemic delivery of vectors, in fully immunocompetent hosts, for a variety of diseases in which delivery of genes may be therapeutically beneficial.

VSV oncolytic virotherapy in the B16 model depends upon intact MyD88 signaling.

We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.

Vesicular stomatitis virus-induced immune suppressor cells generate antagonism between intratumoral oncolytic virus and cyclophosphamide.

Despite having potent oncolytic activity, in vitro, direct intratumoral injection of oncolytic vesicular stomatitis virus (VSV) into established AE17ova mesothelioma tumors in C57Bl/6 mice had no therapeutic effect. During studies to combine systemic cyclophosphamide (CPA) with VSV to suppress the innate immune reaction against VSV, we observed that CPA alone had highly significant antitumor effects in this model. However, against our expectations, the combination of CPA and VSV consistently reduced therapeutic efficacy compared to CPA alone, despite the fact that the combination increased intratumoral VSV titers. We show here that CPA-mediated therapy against AE17ova tumors was immune-mediated and dependent upon both CD4 T cells and natural killer (NK) cells. However, intratumoral VSV induced a transforming growth factor-ß (TGF-ß)-dependent suppressive activity, mediated by CD11b(+)GR-1(+) cells that significantly inhibited both antigen-specific T-cell activation, and CPA-activated, NK-dependent killing of AE17ova tumor cells. Overall, our results show that treatment with oncolytic viruses can induce a variety of immune-mediated consequences in vivo with both positive, or negative, effects on antitumor therapy. These underexplored immune consequences of treatment with oncolytic viruses may have significant, and possibly unexpected, impacts on how virotherapy interacts in combination with other agents which modulate antitumor immune effectors.

Precise scheduling of chemotherapy primes VEGF-producing tumors for successful systemic oncolytic virotherapy.

We have previously reported that a burst of vascular endothelial growth factor (VEGF) signaling to tumor-associated endothelium induces a proviral state, during which systemically delivered oncolytic reovirus can replicate in endothelium, thereby inducing immune-mediated vascular collapse and significant antitumor therapy. Using chimeric receptors, we show here that induction of the proviral state proceeds through VEGFR2, but not VEGFR1, signaling in endothelial cells. In contrast, innate immune activation by reovirus-exposed endothelial cells was predominantly through VEGFR1. By screening conventional chemotherapies for their ability to induce similar effects in combination with reovirus both in vitro and in vivo, we observed that the proviral state could also be induced in endothelial cells exposed to VEGF during rebound from paclitaxel-mediated inhibition of VEGF signaling. We translated these in vitro findings in vivo by careful scheduling of paclitaxel chemotherapy with systemic virotherapy, neither of which alone had therapeutic effects against B16 tumors. Systemic availability of reovirus during endothelial cell recovery from paclitaxel treatment allowed for endothelial replication of the virus, immune-mediated therapy, and tumor cures. Therefore, careful scheduling of combination viro- and chemotherapies, which preclinical testing suggests are individually ineffective against tumor cells, can lead to rational new clinical protocols for systemic treatments with oncolytic viruses.

Synergistic effects of oncolytic reovirus and docetaxel chemotherapy in prostate cancer.

BACKGROUND: Reovirus type 3 Dearing (T3D) has demonstrated oncolytic activity in vitro, in in vivo murine models and in early clinical trials. However the true potential of oncolytic viruses may only be realized fully in combination with other modalities such as chemotherapy, targeted therapy and radiotherapy. In this study, we examine the oncolytic activity of reovirus T3D and chemotherapeutic agents against human prostate cancer cell lines, with particular focus on the highly metastatic cell line PC3 and the chemotherapeutic agent docetaxel. Docetaxel is the standard of care for metastatic prostate cancer and acts by disrupting the normal process of microtubule assembly and disassembly. Reoviruses have been shown to associate with microtubules and may require this association for efficient viral replication. METHODS: The effects of reovirus and chemotherapy on in vitro cytotoxicity were investigated in PC3 and Du 145 cells and the interactions between agents were assessed by combination index analysis. An Annexin V/propidium iodide fluorescence-activated cell sorting-based assay was used to determine mode of cell death. The effects of reovirus and docetaxel administered as single agent or combination therapy were tested in vivo in a murine model. The effects of docetaxel and reovirus, alone and together, on microtubule stabilisation were investigated by Western blot analysis. RESULTS: Variable degrees of synergistic cytotoxicity were observed in PC3 and Du 145 cells exposed to live reovirus and several chemotherapy agents. Combination of reovirus infection with docetaxel exposure led to increased late apoptotic/necrotic cell populations. Reovirus/docetaxel combined therapy led to reduced tumour growth and increased survival in a PC3 tumour bearing mouse model. Microtubule stabilization was enhanced in PC3 cells treated with reovirus/docetaxel combined therapy compared to other reovirus/chemotherapy combinations. CONCLUSIONS: The co-administration of a variety of chemotherapeutic agents with live reovirus was able to enhance cytotoxicity synergistically in vitro. The combination of docetaxel with reovirus also delayed tumour growth and improved survival in vivo. Enhanced microtubule stabilisation following this combination treatment may, in part, explain the mechanism of synergy. These results provide evidence to support the ongoing clinical trials using these agents.

Inactivation of Replication-Competent Vesicular Stomatitis Virus as SARS-CoV-2 Surrogate on Common Surfaces by Disinfectants.

Surface disinfection is part of a larger mitigation strategy to prevent the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus causing coronavirus disease-2019 (COVID-19). Research evaluating the time, nature, and extent of surface disinfection of replication-competent viruses is needed. We evaluated the efficacy of two disinfectants against a replication-competent SARS-CoV-2 surrogate on three common public surfaces. Vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) was our replication-competent SARS-CoV-2 surrogate. Disinfection occurred using Super Sani-Cloth Germicidal Disposable Wipes and Oxivir Tb spray per manufacturer instructions to test the efficacy at reducing the presence, viability, and later replication of VSV-GFP on stainless steel, laminate wood, and porcelain surfaces using standardized methods after recovery and toxicity testing. During the main trials, we placed 100 µL spots of VSV-GFP at viral titers of 108, 107, and 106 PFU/mL on each surface prior to disinfection. Trials were completed in triplicate and post-disinfection measurements on each surface were compared to the measurements of non-disinfected surfaces. Disinfectants were considered efficacious when ≥3-log10 reduction in the number of infectious VSV-GFP virus units was observed on a given surface during all trials. Both disinfectants produced a ≥3.23-log10 reduction in infectious VSV-GFP virus unit numbers, with all trials showing no viable, replication-competent VSV-GFP present on any tested surface. The two disinfectants eliminated the presence, viability, and later replication of VSV-GFP, our SARS-CoV-2 surrogate, on all surfaces. This information suggests that, if following manufacturer instructions, overcleaning surfaces with multiple disinfectant solutions may be unnecessary.

Oncolytic virotherapy induced CSDE1 neo-antigenesis restricts VSV replication but can be targeted by immunotherapy.

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.

Improved systemic delivery of oncolytic reovirus to established tumors using preconditioning with cyclophosphamide-mediated Treg modulation and interleukin-2.

PURPOSE: The goals of this study were (a) to investigate whether preconditioning of immunocompetent mice with PC-61-mediated regulatory T-cell (Treg) depletion and interleukin-2 (IL-2) would enhance systemic delivery of reovirus into subcutaneous tumors and (b) to test whether cyclophosphamide (CPA), which is clinically approved, could mimic PC-61 for modification of Treg activity for translation into the next generation of clinical trials for intravenous delivery of reovirus. EXPERIMENTAL DESIGN: C57Bl/6 mice bearing subcutaneous B16 tumors were treated with CPA or PC-61 followed by 10 injections of low-dose IL-2. Mice were then treated with intravenous reovirus. Virus localization to tumor and other organs was measured along with tumor growth and systemic toxicity. RESULTS: Preconditioning with PC-61 and IL-2 enhanced localization of intravenous oncolytic reovirus to tumors with significantly increased antitumor therapy compared with controls (P < 0.01). However, with the maximal achievable dose of reovirus, Treg modification + IL-2 was also associated with systemic toxicity. CPA (100 mg/kg) did not deplete, but did functionally inhibit, Treg. CPA also mimicked PC-61, in combination with IL-2, by inducing "hyperactivated" NK cells. Consistent with this, preconditioning with CPA + IL-2 enhanced therapy of intravenously delivered, intermediate-dose reovirus to a level indistinguishable from that induced by PC-61 + IL-2, without any detectable toxicity. CONCLUSION: With careful reference to ongoing clinical trials with dose escalation of reovirus alone and in combination with CPA, we propose that future clinical trials of CPA + IL-2 + reovirus will allow for both improved levels of virus delivery and increased antitumor efficacy.

Cell carriers for oncolytic viruses: Fed Ex for cancer therapy.

Oncolytic viruses delivered directly into the circulation face many hazards that impede their localization to, and infection of, metastatic tumors. Such barriers to systemic delivery could be overcome if couriers, which confer both protection, and tumor localization, to their viral cargoes, could be found. Several preclincal studies have shown that viruses can be loaded into, or onto, different types of cells without losing the biological activity of either virus or cell carrier. Importantly, such loading can significantly protect the viruses from immune-mediated virus-neutralizing activities, including antiviral antibody. Moreover, an impressive portfolio of cellular vehicles, which have some degree of tropism for tumor cells themselves, or for the biological properties associated with the tumor stroma, is already available. Therefore, it will soon be possible to initiate clinical protocols to test the hypopthesis that cell-mediated delivery can permit efficient shipping of oncolytic viruses from the loading bay (the production laboratory) directly to the tumor in immune-competent patients with metastatic disease.

Ad-CD40L mobilizes CD4 T cells for the treatment of brainstem tumors.

BACKGROUND: Diffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem. METHODS: Syngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient-derived diffuse midline gliomas and immunocompetent models. RESULTS: Expression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1ß, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1ß, generated significant numbers of cures with readily manageable toxicity. CONCLUSIONS: Virus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.

APOBEC3B-mediated corruption of the tumor cell immunopeptidome induces heteroclitic neoepitopes for cancer immunotherapy.

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.