Kevin G. Shim

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.

Combination viroimmunotherapy with checkpoint inhibition to treat glioma, based on location-specific tumor profiling.

BACKGROUND Systemic delivery of a complementary cDNA library expressed from the vesicular stomatitis virus (VSV) treats tumors by vaccinating against a wide range of tumor associated antigens (TAAs). For subcutaneous B16 melanomas, therapy was achieved using a specific combination of self-TAAs (neuroblastoma-Ras, cytochrome c, and tyrosinase-related protein 1) expressed from VSV. However, for intracranial B16 tumors, a different combination was therapeutic (consisting of VSV-expressed hypoxia-inducible factor [HIF]-2α, Sox-10, c-Myc, and tyrosinase-related protein 1). Therefore, we tested the hypothesis that tumors of different histological types growing in the brain share a common immunogenic signature which can be exploited for immunotherapy. METHODS Syngeneic tumors, including GL261 gliomas, in the brains of immune competent mice were analyzed for their antigenic profiles or were treated with systemic viroimmunotherapy. RESULTS Several different histological types of tumors growing intracranially, as well as freshly resected human brain tumor explants, expressed a HIF-2α(Hi) phenotype imposed by brain-derived CD11b+ cells. This location-specific antigen expression was exploited therapeutically against intracranial GL261 gliomas using systemically delivered VSV expressing HIF-2α, Sox-10, and c-Myc. Viroimmunotherapy was enhanced by immune checkpoint inhibitors, associated with the de-repression of antitumor T-helper cell type 1 (Th1) interferon-γ and Th17 T cell responses. CONCLUSIONS Since different tumor types growing in the same location in the brain share a location-specific phenotype, we suggest that antigen-specific immunotherapies should be based upon expression of both histological type-specific tumor antigens and location-specific antigens. Our findings support clinical application of VSV-TAA therapy with checkpoint inhibition for aggressive brain tumors and highlight the importance of the intracranial microenvironment in sculpting a location-specific profile of tumor antigen expression.

Definitive Management of Oligometastatic Melanoma in a Murine Model Using Combined Ablative Radiation Therapy and Viral Immunotherapy.

PURPOSE The oligometastatic state is an intermediate state between a malignancy that can be completely eradicated with conventional modalities and one in which a palliative approach is undertaken. Clinically, high rates of local tumor control are possible with stereotactic ablative radiation therapy (SABR), using precisely targeted, high-dose, low-fraction radiation therapy. However, in oligometastatic melanoma, virtually all patients develop progression systemically at sites not initially treated with ablative radiation therapy that cannot be managed with conventional chemotherapy and immunotherapy. We have demonstrated in mice that intravenous administration of vesicular stomatitis virus (VSV) expressing defined tumor-associated antigens (TAAs) generates systemic immune responses capable of clearing established tumors. Therefore, in the present preclinical study, we tested whether the combination of systemic VSV-mediated antigen delivery and SABR would be effective against oligometastatic disease. METHODS AND MATERIALS We generated a model of oligometastatic melanoma in C57BL/6 immunocompetent mice and then used a combination of SABR and systemically administered VSV-TAA viral immunotherapy to treat both local and systemic disease. RESULTS Our data showed that SABR generates excellent control or cure of local, clinically detectable, and accessible tumor through direct cell ablation. Also, the immunotherapeutic activity of systemically administered VSV-TAA generated T-cell responses that cleared subclinical metastatic tumors. We also showed that SABR induced weak T-cell-mediated tumor responses, which, particularly if boosted by VSV-TAA, might contribute to control of local and systemic disease. In addition, VSV-TAA therapy alone had significant effects on control of both local and metastatic tumors. CONCLUSIONS We have shown in the present preliminary murine study using a single tumor model that this approach represents an effective, complementary combination therapy model that addresses the need for both systemic and local control in oligometastatic melanoma.

Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence.

Patients with minimal residual disease are at high risk for relapse. The authors now show how innate immune surveillance mechanisms that normally control infection and the growth of primary tumors are subverted by residual disease to promote recurrence. Understanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment-resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immunosurveillance. In the first, the role of TNFα changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)–mediated cytokine response characterized by low IL6 and elevated IFNγ, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNγ, inhibiting both NK-cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFα, which stimulated the growth of MRD tumors. Finally, therapies that blocked PD-1, TNFα, or NK cells delayed or prevented recurrence. These data show how innate immunosurveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identify therapeutic targets in patients with MRD known to be at high risk of relapse. Cancer Immunol Res; 5(11); 1029–45. ©2017 AACR.

Mutated BRAF emerges as a major effector of recurrence in a murine melanoma model after treatment with immunomodulatory agents

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595-605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence.

Ad5NULL-A20: A Tropism-Modified, αvβ6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues. Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vectors selectivity in vitro and in vivo was assessed. Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20–treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215–24. ©2018 AACR.

69. Combination Therapy of Reovirus and PD-1 Blockade Effectively Establishes Tumor Control Via Innate and Adaptive Immune Responses

Combination therapy of Reovirus and PD-1 blockade effectively establishes tumor control via innate and adaptive immune responsesKarishma Rajani1, Christopher Parrish2, Kevin Shim1, Liz Ilett2, Jill Thompson1, Tim Kottke1, Jose Pulido1, Fiona Errington-Mais2, Peter Selby2, Hardev Pandha3, Kevin Harrington4, Alan Melcher2, Rosa Maria Diaz1, Shane Zaidi1,4 Matt Coffey5, and Richard Vile1,2,6Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA. 2Leeds Institute of Cancer and Pathology, St. JamesUniversity Hospital, Leeds, UK. 3University of Surrey, Guildford, UK. 4. The Institute of Cancer Research, 237 Fulham Road, London, SW3.5Oncolytics Biotech Inc., Calgary, Canada. 6Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA.Reovirus has oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. We have shown that anti-tumor therapy is directly associated with immune activation by virus replication in tumors. The immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, as well as the generation of adaptive anti tumor immune responses as a result of in vivo priming against tumor associated antigens released during that killing. To exploit the immune components of reovirus anti-tumor therapy, we hypothesized that the combination of reovirus therapy with systemic checkpoint inhibition would augment therapeutic efficacy. To establish this, subcutaneous (SC) B16 melanomas were treated with reovirus alone or in combination with antibody against PD-1. In this model, intra-tumoral (IT) injection of reovirus into SC tumors generated moderate therapy. Systemic treatment of anti-PD-1 antibody along with IT reovirus, significantly enhanced survival compared to IT reovirus alone (p 40% of mice being cured long term. Immune analysis suggested that the enhanced therapeutic benefit of reovirus plus checkpoint inhibition is contributed by at least two factors. First, blockade of PD-1 significantly enhanced the ability of NK cells to recognize (TNF-α secretion), and kill, reovirus-infected tumor cells. Second, anti PD-1 antibody led to a significant reduction in Treg activity in reovirus-treated mice, with the overall effect of increasing the adaptive CD8+ anti-tumor T cell response. In vivo depletion studies demonstrated that NK cells had a dramatic effect in reducing the therapeutic efficacy of reovirus plus anti-PD-1 therapy. These results indicate that combination therapy of reovirus with PD-1 blockade confers significant survival benefit, by augmenting tumor-specific NK responses and attenuating tumor-specific immunosuppression and is a viable treatment modality with far greater efficacy than either therapy alone.

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.

64. Generation of Tumor Cells Resistant to Oncolysis Is Mediated Through Virus Induced APOBEC Expression

In both pre-clinical, and clinical models, we have observed that treatment of primary tumors with oncolytic viruses can lead to very significant tumor regressions, often with apparent disappearance of the tumor. However, in many cases, tumor subsequently recurs, often extremely aggressively. We have been able to mimic this effect in vitro by growing several different types of tumor cell lines in the presence of different oncolytic viruses at low m.o.i. over several weeks. Under such conditions, we consistently observe the emergence of cells which survive over long periods of time in culture, despite the demonstrable presence of ongoing viral replication. We identified APOBEC3 from a screen of genes which are induced in tumor cells undergoing continual exposure to either reovirus or Vesicular Stomatitis Virus (VSV). In this respect, overexpression of APOBEC3B, a cytidine deaminase, has been identified in human tumors associated with mutations that may drive tumorigenesis. Therefore, we tested the hypothesis that, upon infection with oncolytic viruses, APOBEC3 may help drive tumor cell mutation leading to protection from viral cytotoxicity. Consistent with this, both mRNA and protein levels of APOBEC3 were rapidly induced within 24-72 hours of low M.O.I infection by reovirus, or VSV, of B16 melanoma, GL261 glioma and TC2 prostate tumor murine cell lines. Similar low level infection of human tumor cell lines was associated with rapid induction of the human APOBEC3B mRNA. Interestingly, engineered over-expression of APOBEC3B in tumor lines significantly enhanced the ability of these cells to resist killing by either VSV or reovirus. This effect was inhibited by blockade of PKC signaling upon viral infection but was enhanced by the presence of type I interferons. Correspondingly, inhibition of APOBEC3 using shRNA decreased the frequency of emergence of VSV-resistant tumor populations. These data suggest that infection of tumor cells by oncolytic viruses at low M.O.I (as is likely to be the case during clinical treatments) leads to the induction of cellular proteins, which enhance the ability of resistance to oncolysis to develop. Deep sequencing studies are currently underway to determine the genetic changes in both virus, and target tumor cells, which are associated with APOBEC3 over-expression during chronic exposure to oncolytic virus infection. Finally, data will be presented on how these findings allow the construction of improved viruses for cancer therapy by targeting APOBEC3 induction to improve primary killing and prevent emergence of treatment resistant populations.

APOBEC3 Mediates Resistance to Oncolytic Viral Therapy

Tumor cells frequently evade applied therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor that restricts the potency of oncolytic vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFN-β-dependent manner, which was responsible for the evolution of virus-resistant cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. Knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV both in vitro and in vivo. Finally, we demonstrate that APOBEC3B expression had a direct effect on the fitness of VSV, an RNA virus that has not previously been identified as restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of viral or broader nucleic acid-based therapeutic platforms.