Citations

Abstract We have previously shown that a vesicular stomatitis virus-based cDNA library expressing xenogeneic altered self-epitopes led to T-cell mediated rejection of prostate cancer, glioma and melanoma. The use of a library of altered self-peptides which reflects the transcriptome of the tumor circumvents the necessity for a priori knowledge of which antigens may be immunogenic in a patient, and primes a polyclonal T-cell response that provides strong cumulative antitumor selective pressure. We have expanded this concept of altered-self library vaccination using tumor vaccines modified by the overexpression of APOBEC3B, a cytosine deaminase that generates C to T transition mutations. A freeze-thawed whole tumor cell vaccine prepared from B16 melanoma cells stably overexpressing human APOBEC3B treated established subcutaneous parental B16 tumors and, when combined with PD-1 checkpoint blockade, cured between 75%-100% of mice. T-cells from mice treated with the vaccine and anti-PD1 produced high levels of IFN-γ when restimulated with parental unmodified B16 melanoma cells in vitro. Whole-genome sequencing of B16APOBEC3B overexpressing cells identified 301 C to T or G to A missense mutations unique to the APOBEC3B line. Using an in silico MHC binding affinity algorithm, we identified and experimentally validated a short list of 10 APOBEC3B- induced heteroclitic peptides. We have also shown that this approach can be extended to an orthotopic brainstem model of high grade glioma. GL261 tumors stereotactically implanted into the brainstem were significantly responsive to treatment with an APOBEC3B- modified GL261 vaccine in combination with anti-PD-1 checkpoint blockade. In summary, when overexpressed in tumor cells, APOBEC3B generates a library of heteroclitic sequences that primes both CD4 and CD8 T-cells that have escaped central tolerance and that recognize both newly mutated antigens from the vaccine, and the corresponding unaltered self epitopes expressed on the tumor cells. Citation Format: Richard Vile, Laura Evgin, Timothy Kottke, Matthew Schuelke, Christopher B. Driscoll, Amanda L. Huff, Jill Thompson, Amy Molan, Reuben S. Harris, Jose S. Pulido, Phonphimon Wongthida. Cancer immunotherapy with APOBEC3B-induced heteroclitic library tumor cell vaccines and immune checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B138.

Abstract Tumor cells are able to evade cytotoxic 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 involved in the restriction of vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFNβ-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. ShRNA 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 in murine melanoma and glioblastoma lines, as well as in a human melanoma cell line in vitro and in vivo. Finally, we demonstrate that progeny virus obtained after passage through APOBEC3B overexpressing cells contained more defective interfering particles, thereby indicating that APOBEC3B directly affected the fitness of VSV, an RNA virus that has not previously been identified to be restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of oncolytic viruses as well as broader nucleic acid-based therapeutic platforms. Citation Format: Amanda L. Huff, Phonphimon Wongthida, Timothy Kottke, Jill Thompson, Christopher B. Driscoll, Matthew Schuelke, Kevin G. Shim, Reuben S. Harris, Amy Molan, Jose S. Pulido, Peter J. Selby, Kevin J. Harrington, Alan Melcher, Laura Evgin, Richard Vile. APOBEC3 confers resistance to oncolytic VSV therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A133.

Abstract Although the chimeric antigen receptor (CAR) T-cell platform has experienced clinical success in patients with hematologic malignancies, CAR T-cells specific to solid tumor targets have met with more limited efficacy. The highly inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment suggested to us that they would provide a complementary mechanism of action to both recruit and potentiate the functionality of CAR T-cells. VSVmIFNβ injection into B16EGFRvIII tumors increased the expression of chemokines such as CXCL10 and CCL5, which we would expect to recruit CXCR3+and CCR5+ EGFRvIII specific murine CAR T-cells. However, we did not observe an increase in overall survival or tumor control using the combination strategy compared to monotherapy with CAR T-cells. We recovered fewer viable CD8+ CAR T-cells from tumors injected with VSVmIFNβ early after adoptive transfer, and observed a similar reduction in the number of CD8+ CAR T-cells which persisted long term in the blood. We have shown that type I interferon increases the expression of the CAR from the retroviral LTR and in turn sensitizes these cells to tonic signaling mediated exhaustion and apoptosis. Correspondingly, CAR T-cells prepared from IFNAR KO T-cells were protected from the deleterious effect of type I IFN in vivo. We are currently investigating strategies to overcome the interference between these two modalities and to uncouple the regulation of the expression of the CAR from type I IFN signaling. Citation Format: Laura Evgin, Amanda L. Huff, Phonphimon Wongthida, Jill Thompson, Timothy Kottke, John Sampson, Luis Sanchez Perez, Richard Vile. Unexpected antagonism between oncolytic virus derived type I interferon and EGFRvIII CAR T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A029.

Abstract Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus–antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161–73. ©2018 AACR.

Abstract 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 vector's 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.

Intravenous infusion of oncolytic reovirus in patients leads to infection of brain tumors, infiltration by cytotoxic T cells, and up-regulation of PD-L1.

AbstractUnderstanding 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.

Abstract We have developed the use of reovirus as a systemically delivered oncolytic agent in both pre-clinical models and in Phase I and II clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. In addition however, 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. Therefore, to exploit the immune components of reovirus anti-tumor therapy, we tested the combination of oncolytic therapy with systemic checkpoint inhibition. To do this, we used our murine, immune-competent, model and a protocol in which injection of reovirus into subcutaneous (s.c.) B16 melanomas generates moderate therapy following intra-tumoral injection of reovirus into 5d established subcutaneous B16 melanomas growing in C57Bl/6 mice. In this model, provision of systemic anti-PD-1 antibody along with i.t. reovirus, significantly enhanced survival compared to i.t. reovirus alone (p<0.01) and led to >40% of mice being cured long term. Immune analysis suggests that the enhanced therapeutic benefit of reovirus plus checkpoint inhibition is contributed by at least two factors. Thus, blockade of PD-1 significantly enhanced the ability of NK cells to recognize (TNF-α secretion), and kill, reovirus-infected target tumor cells. Second, antiPD-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. We also showed that timing of the checkpoint inhibitor antibody was significant in balancing toxicity with anti tumor therapy. These data suggest that combination of checkpoint inhibition therapy with reovirus oncolytic/immunotherapy represents a readily translatable method to enhance the therapeutic value of either alone. Citation Format: Shane Zaidi, Kevin Shim, Karishma Rajani, Rosa Diaz, Elizabeth Ilett, Timothy Kottke, Jill Thompson, Peter Selby, Kevin Harrington, Hardev Pandha, Alan Melcher, Matt Coffey, Richard Vile. Combination therapy of reovirus and checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B88.

Abstract We have developed the use of reovirus as a systemically delivered oncolytic agent in both pre-clinical models and in early Phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. In addition however, 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. Therefore, 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 test the hypothesis, we used C57Bl/6 mice, an immune-competent murine model, with established subcutaneous (s.c.) B16 melanomas. In this model, intra-tumoral injection of reovirus into s.c. tumors generated moderate therapy. Provision of systemic anti-PD-1 antibody along with i.t. reovirus, significantly enhanced survival compared to i.t. reovirus alone (p<0.01) and led to >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 target 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. Furthermore, in vivo depletion studies demonstrated that NK cells had a dramatic effect in reducing the therapeutic efficacy of reovirus plus anti-PD-1 therapy. Overall, the results indicate that combination therapy of reovirus with PD-1 blockade confers significant survival benefit, by augmenting tumor-specific NK responses and specifically attenuating tumor-specific immunosuppression. These data also suggest that combination of PD-1 inhibition therapy with reovirus oncolytic/immunotherapy represents a readily translatable method to enhance the therapeutic efficacy. Citation Format: Karishma Rajani, Christopher Parrish, Kevin Shim, Liz Ilett, Fiona Errington-Mais, Jill Thompson, Tim Kottke, Rosa Maria-Diaz, Peter Selby, Hardev Pandha, Kevin Harrington, Alan Melcher, Matt Coffey, Shane Zaidi, Richard Vile. Combination therapy of reovirus and PD-1 blockade effectively establishes tumor control via innate and adaptive immune responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1360. doi:10.1158/1538-7445.AM2015-1360