Díaz, Rosa María

Abstract Introduction Voyager-V1TM is derived from VSV, a bullet-shaped negative sense RNA virus with very low human seroprevalence; it is engineered to selectively replicate in and kill human cancer cells. Voyager-V1 encodes the human IFNβ gene to boost antitumoral immune responses and the thyroidal sodium iodide symporter NIS gene to permit noninvasive imaging of virus spread. FIH studies are underway via both IV and IT routes. Here we report safety and preliminary correlative data from the FIH IT study. Methods and objectives This is a classical 3+3 phase 1 design, using escalating single IT viral doses from 3 x 106 to 3 x 109 TCID50 into one target lesion. The primary objective is safety and tolerability, monitored by committee. Other objectives include PK by RT-PCR for viral genomes, serum IFNβ levels, Tc-99m SPECT/CT imaging to monitor virus infection in injected lesions, peripheral blood immunophenotyping with 11-color flow cytometry for activation markers on T cells, T-regs, NK cells, and MDSCs, and serial biopsies to assess the tumor microenvironment (TME). IHC was performed on tumor biopsies for CD3, CD8, CD4, FoxP3, CD68, PD-1 and PDL-1 pre and post treatment in non-injected and injected lesions. CD45 staining is ongoing. Results Dose level 3 is ongoing (n=8+). No DLTs have been observed to date. Most patients were male (75%), white (100%), with ECOG PS1 (75%) and a median of >6 lines of prior systemic therapy. AEs (in 63% patients) reported as related to study drug were mild-moderate, short-lived, and consisted of G1 fever, chills, hot flashes, nausea, vomiting, hyperhidrosis and G2 fatigue. Most patients had some mild AEs (pain, bruising, subclinical pneumothorax) related to biopsy and IT injection procedures and one had an SAE related to biopsy (G2 pneumothorax). There was no difference in incidence, intensity or duration of related AEs between dose levels so far. In patients analyzed to date, at the first three dose levels of 3 x 106, 1 x 107 and 3 x 107 TCID50 (n=7), viremia and serum IFNβ were below levels of detection after IT injection. There is evidence in all patients of an increase in PD-1 expression on CD4 and CD8 T cells, suggesting T cell activation post- single virus injection. One patient at dose level 2 had increased CD4 and CD8 T cells in the peripheral blood. TME analysis revealed increased infiltration of CD3 and CD8 cells in tumors of some patients. In addition, there are changes in numbers of FoxP3 and CD68 positive cells in the injected lesions compared to the pre-treatment and non-injected lesion. Two patients at dose level 3, one with an injected adrenal metastasis of colorectal cancer and another with a chest wall lesion from a head of pancreas primary, have positive SPECT/CTs on days 3-15 showing viral replication in tumor plus concomitant lymphocyte/neutrophil trafficking in the periphery. Conclusions IT injection of a single dose of the novel oncolytic virotherapy Voyager-V1 has proven safe at doses up to 3 x 107 TCID50. There was evidence of T cell activation with increased PD-1 expression in peripheral blood lymphocytes after a single IT injection. Tumor biopsies indicate increased lymphocyte infiltration. Two patients at the highest dose tested show signs of viral replication in the injected lesion plus evidence of systemic impact on leukocyte populations. Citation Format: Steven Powell, Manish R. Patel, Jaime R. Merchan, Timothy P. Cripe, James Strauss, Rosa M. Diaz, Nandakumar Packiriswamy, Bethany A. Brunton, Deepak Upreti, Rehan Khan, Lukkana Suksanpaisan, Rianna Vandergaast, Stephen J. Russell, Alice S. Bexon, Kah Whye Peng. Preliminary correlative and clinical data from a first-in-human (FIH) study of the intratumoral (IT) oncolytic virotherapy, Voyager-V1, in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT051.

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 Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-α. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.

Abstract Oncolytic virotherapy offers the potential to treat tumors both as a single agent and in combination with traditional modalities such as chemotherapy and radiotherapy. Here we describe an effective, fully systemic treatment regimen, which combines virotherapy, acting essentially as an adjuvant immunotherapy, with adoptive cell transfer (ACT). The combination of ACT with systemic administration of a vesicular stomatitis virus (VSV) engineered to express the endogenous melanocyte antigen glycoprotein 100 (gp100) resulted in regression of established melanomas and generation of antitumor immunity. Tumor response was associated with in vivo T-cell persistence and activation as well as treatment-related vitiligo. However, in a proportion of treated mice, initial tumor regressions were followed by recurrences. Therapy was further enhanced by targeting an additional tumor antigen with the VSV-antigen + ACT combination strategy, leading to sustained response in 100% of mice. Together, our findings suggest that systemic virotherapy combined with antigen-expressing VSV could be used to support and enhance clinical immunotherapy protocols with adoptive T-cell transfer, which are already used in the clinic. Cancer Res; 72(18); 4753–64. ©2012 AACR.

An antitumor T-cell response can lead to tumor control without clearing all tumor cells. As long as residual tumor cells remain, there is a constant risk of escape from that T-cell response. We previously showed that adoptive transfer of anti-ova OT-I T cells into B16ova-bearing mice led to tumor regression followed by escape of tumors that had lost the ova gene, rendering the OT-I T cells ineffective. In this study, we hypothesized that simultaneous transfer of cytotoxic T lymphocytes targeted against two independent antigens would reduce selection for single-antigen-loss cells, thereby limiting tumor escape. Using OT-I and Pmel T cells to treat B16ova tumors, we found that early cotransfer could prevent tumor emergence in most mice, whereas neither T-cell specificity alone was able to do so. When combined with total body irradiation for the treatment of larger 7-day tumors, cotransfer was also better at limiting tumor recurrence, and the tumors that did escape combination therapy continued to express both target antigens. As adoptively transferred T cells also persisted in vivo , even in mice with recurrent tumors, we hypothesized that restimulation of these antitumor T cells would prolong survival of mice with recurrent tumors. Consistent with this hypothesis, administration of a low-dose regimen of cyclophosphamide following tumor escape slowed tumor growth in mice that had previously received T-cell therapy, but not in control-treated mice, an effect that was associated with increased activation of T cells in vitro by low- but not high-dose cyclophosphamide.

Abstract Innate immune effector mechanisms triggered by oncolytic viruses may contribute to the clearance of both infected and uninfected tumor cells in immunocompetent murine hosts. Here, we developed an in vitro tumor cell/bone marrow coculture assay and used it to dissect innate immune sensor and effector responses to intratumoral vesicular stomatitis virus (VSV). We found that the type III IFN interleukin-28 (IL-28) was induced by viral activation of innate immune-sensing cells, acting as a key mediator of VSV-mediated virotherapy of B16ova melanomas. Using tumor variants which differentially express the IL-28 receptor, we showed that IL-28 induced by VSV within the tumor microenvironment sensitizes tumor cells to natural killer cell recognition and activation. These results revealed new insights into the immunovirological mechanisms associated with oncolytic virotherapy in immune-competent hosts. Moreover, they defined a new class of tumor-associated mutation, such as acquired loss of responsiveness to IL-28 signaling, which confers insensitivity to oncolytic virotherapy through a mechanism independent of viral replication in vitro. Lastly, the findings suggested new strategies to manipulate immune signals that may enhance viral replication, along with antitumor immune activation, and improve the efficacy of oncolytic virotherapies. Cancer Res; 70(11); 4539–49. ©2010 AACR.

Abstract Our preclinical and clinical trials using a replication-defective adenoviral vector expressing IFN-β have shown promising results for the treatment of malignant mesothelioma. Based on the hypotheses that a replication-competent vesicular stomatitis virus (VSV) oncolytic vector would transduce more tumor cells in vivo, that coexpression of the immunostimulatory IFN-β gene would enhance the immune-based effector mechanisms associated both with regression of mesotheliomas and with VSV-mediated virotherapy, and that virus-derived IFN-β would add further safety to the VSV platform, we tested the use of IFN-β as a therapeutic transgene expressed from VSV as a novel treatment for mesothelioma. VSV-IFN-β showed significant therapy against AB12 murine mesotheliomas in the context of both local and locoregional viral delivery. Biologically active IFN-β expressed from VSV added significantly to therapy compared with VSV alone, dependent in part on host CD8+ T-cell responses. Immune monitoring suggested that these antitumor T-cell responses may be due to a generalized T-cell activation rather than the priming of tumor antigen–specific T-cell responses. Finally, IFN-β also added considerable extra safety to the virus by providing protection from off-target viral replication in nontumor tissues and protected severe combined immunodeficient mice from developing lethal neurotoxicity. The enhanced therapeutic index provided by the addition of IFN-β to VSV therefore provides a powerful justification for the development of this virus for future clinical trials. [Cancer Res 2009;69(19):7713–20]