Tim Kottke

Immune-Mediated Antitumor Activity of Reovirus Is Required for Therapy and Is Independent of Direct Viral Oncolysis and Replication

Purpose: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. Experimental Design: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. Results: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. Conclusion: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.

A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines

Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell–DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.

Allogeneic tumor cells expressing fusogenic membrane glycoproteins as a platform for clinical cancer immunotherapy.

PURPOSE: Fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus G glycoprotein (VSV-G), represent a new class of gene therapy for cancer that cause cytotoxic fusion on expression in tumor cells. In addition, FMG-mediated tumor cell death stimulates antitumor immunity, suggesting potential applications for FMG-expressing cellular vaccines. This study addresses the promise of FMG-expressing allogeneic tumor cells, which are most practical for clinical use, as a novel platform for ex vivo and in situ vaccination. EXPERIMENTAL DESIGN: Murine B16 melanoma-derived cell lines expressing autologous or allogeneic MHC class I, expressing fusogenic or nonfusogenic VSV-G, were used to vaccinate mice in vivo against a live tumor challenge. Exosome-like vesicles released by fusing allogeneic cells (syncitiosomes) and intratumoral injection of fusing vaccines were also tested as novel therapeutic strategies for their antitumor effects. RESULTS: Expression of fusogenic VSV-G enhanced the immunogenicity of an allogeneic cellular vaccine, which was more effective than a fusing autologous vaccine. Allogeneic syncitiosomes were only as effective as cellular vaccines when administered with adjuvant, demonstrating that syncitiosomes cannot account entirely for the mechanism of immune priming. Intratumoral injection of FMG-expressing allogeneic cells led to significant tumor regression using both fusogenic or nonfusogenic VSV-G. However, specific priming against tumor-associated antigenic epitopes and protection against secondary rechallenge only occurred if the initial vaccine was competent for cell fusion. CONCLUSIONS: FMG-expressing allogeneic tumor cells are a potent source of antitumor vaccines. Syncitiosomes given with adjuvant and intratumoral injection of fusing cells represent novel strategies well-suited to clinical translation.

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 Txa0cells (adoptive cell therapy, ACT) inxa0vivo 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+ Txa0cells 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 Txa0cells and the acute Txa0cell response to oncolytic viruses.

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.

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.

749. Escape from Tumor Dormancy Following Gene- or Viro-Therapies Is Mediated by Acquisition of a Phenotype in Which Innate Immune Surveillance Actively Drives Tumor Cell Recurrence

When established (~0.3-0.5cm) primary murine B16 tumors are treated with either cytotoxic gene therapy (HSVtk/GCV), oncolytic virotherapy (intra-tumoral reovirus) or adoptive T cell therapy, a proportion of mice will become apparently tumor free. However, if the animals are observed for prolonged periods of time (>60-100 days), the majority of mice will develop aggressively growing recurrent tumors. Excision of the site of tumor injection during the period of tumor dormancy (no palpable tumor) indicated that residual tumor cells are readily detectable in most mice by histology. Unexpectedly, it was extremely difficult to re-grow the tumor cells from these specks of minimal residual disease in vitro. However, by screening multiple cytokines for their ability to support re-growth of recurrent B16 cells, we identified TNF-α as a major growth factor for recurrent cells. Conversely, treatment of primary B16 populations with TNF-α led to highly significant reductions in tumor cell viability. Similarly, depletion of NK cells, or antibody blockade of TNF-α, from C57Bl/6 mice led to significantly increased tumorigencity of primary B16 tumors. In contrast, depletion of NK cells allowed for decreased tumorigenicity of TNF-α stimulated recurrent B16 cells recovered from mice in a state of minimal residual disease. These data suggest that tumor recurrence may be mediated by a distinct phenotypic switch in vivo. Thus, initially, primary tumor cells are highly sensitive to innate immune cell surveillance through NK cells and TNF-α. However, escape from dormancy in vivo is associated with acquisition of a phenotype in which such TNF-α-mediated immune surveillance actively drives tumor growth. TNF-α growth promoted recurrent cells also expressed high levels of PD-L1 compared to primary tumor cells, suggesting that this may be a further mechanism by which recurrences could emerge in vivo, even in the presence of anti tumor T cell responses. Consistent with this hypothesis, recurrence was significantly inhibited in vivo following gene, viro- or adoptive T cell therapies, by treatment with TNF-α blockade or systemic checkpoint inhibitor therapy.

63. Immunogenicity of Self Tumor Associated Antigens Is Enhanced Through Protein Truncation

We showed previously that expressing tumor-associated antigens (TAA) from Vesicular Stomatitis Virus (VSV) eradicates established tumors. We show here that truncation of TAA expressed from VSV can occur to preserve the ability of the virus to replicate efficiently. We observed that truncation of VSV-expressed TAA affects the processing of the antigen, causing a bias towards an IL-17 anti-tumor response which was raised by cumulative signaling from different types of APC, each presenting specific, truncated antigens. Whereas processing of full length, self-TAA invoked an IFN-γ based, CD8+ dependent response, truncated versions of the same self-TAA (likely to be poorly and incompletely folded) were processed through a class II-dependent pathway, and invoked an IL-17 based response. Significantly, the IL-17-mediated anti tumor response was both more therapeutic, and durable, than the response against full length self-TAA. These data show that the type/potency of anti-tumor immune responses against self-TAA can be manipulated in vivo through the nature of the self protein (full length or truncated), inclusion of multiple TAA to recruit the optimal combination of APC, and the resultant skewing of the T cell response to either an IFN-γ or IL-17 producing phenotype. Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-TAA can be broken through manipulation of protein integrity, allowing for rational design of better self immunogens for cancer immunotherapy.