Thompson, Jill

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]

Abstract We have a long-term interest in the connectivity between autoimmunity and tumor rejection. However, outside of the melanocyte/melanoma paradigm, little is known about whether autoimmune responses to normal tissue can induce rejection of tumors of the same histologic type. Here, we induced direct, pathogen-like cytotoxicity to the normal pancreas in association with the immune adjuvant heat shock protein 70. In sharp contrast to our studies with a similar approach for the treatment of prostate cancer, inflammatory killing of the normal pancreas induced a Th1-like, anti-self-response to pancreatic antigens, which was rapidly suppressed by a concomitant suppressive regulatory T cell (Treg) response. Interestingly, even when Treg cells were depleted, the Th1-like response was insufficient to induce significant ongoing autoimmunity. However, the Th1-like response to antigens expressed in the pancreas at the time of damage was sufficient to induce rejection of tumors expressing either a foreign (ova) antigen or fully syngeneic tumor antigens (on Panc02 tumor cells), provided that Treg were depleted before inflammatory killing of the normal pancreas. Taken together, these data indicate that profound differences exist between the immunoprotective mechanisms in place between different tissues (pancreas and prostate) in their response to pathogen-like damage. Moreover, they also show that, although multiple layers of immunologic safeguards are in place to prevent the development of severe autoimmune consequences in the pancreas (in contrast to the prostate), tumor rejection responses can still be decoupled from pathologic autoimmune responses in vivo, which may provide novel insights into the immunotherapeutic treatment of pancreatic cancer. [Cancer Res 2009;69(19):7767–74]

Abstract 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.

Abstract 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.

AbstractPurpose: Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity.Experimental Design: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated.Results: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-γ production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8+ T cells specific against the human tumor-associated antigen MART-1.Conclusion: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.

Abstract Purpose: The purpose of the present study was to investigate whether it is possible to achieve truly systemic delivery of oncolytic reovirus, in immunocompetent hosts, using cyclophosphamide to overcome some of the barriers to effective intratumoral delivery and replication of i.v. injected virus. Experimental Design: I.v. delivery of reovirus was combined with different regimens of i.p. administered cyclophosphamide in C57Bl/6 mice bearing established s.c. B16 tumors. Intratumoral viral replication, tumor size, and survival were measured along with levels of neutralizing antibody (NAb) in the blood. Finally, differential toxicities of the virus/cyclophosphamide regimens were monitored through viral replication in systemic organs, survival, and cardiac damage. Results: Repeated i.v. injection of reovirus was poorly effective at seeding intratumoral viral replication/oncolysis. However, by combining i.v. virus with cyclophosphamide, viral titers of between 107 and 108 plaque-forming units per milligram were recovered from regressing tumors. Doses of cyclophosphamide that ablated NAb were associated with severe toxicities, characterized by viral replication in systemic organs—toxicities that are mirrored by repeated reovirus injections into B-cell knockout mice. Next, we restructured the dosing of cyclophosphamide and i.v. virus such that a dose of 3 mg cyclophosphamide was administered 24 h before reovirus injection, and this schedule was repeated every 6 days. Using this protocol, high levels of intratumoral viral access and replication (∼107 plaque-forming units per milligram tumor) were maintained along with systemically protective levels of NAb and only very mild, non–life-threatening toxicity. Conclusion: NAb to oncolytic viruses play a dual role in the context of systemic viral delivery; on one hand, they hinder repeated administration of virus but on the other, they provide an important safety mechanism by which virus released from vigorous intratumoral replication is neutralized before it can disseminate and cause toxicity. These data support the use of cyclophosphamide to modulate, but not ablate, patient NAb, in development of carefully controlled clinical trials of the systemic administration of oncolytic viruses.

AbstractA close connectivity between autoimmune and tumor rejection responses is known to exist in the case of melanoma immunotherapy. However, relatively little is known about self-antigens on other types of normal cells, their relation to the development of autoimmune disease, and their possible coexistence as potential tumor rejection antigens on associated tumors. In the current study, we induced inflammatory killing of normal prostate tissue in situ using a fusogenic membrane glycoprotein along with the immune adjuvant hsp70. We show here that, in the prostate, hsp70 induces interleukin (IL)-6, which triggers a CD4- and CD8-dependent progressive autoimmune reactivity, associated with IL-17 expression. This autoimmune response was also able to induce the rejection of established prostate tumors, but not other histologic types of tumors, growing elsewhere in the animal. These data show that the intimate connectivity between autoimmune and tumor rejection responses extends beyond the classic melanoma paradigm and may be clinically valuable for the treatment of established metastatic disease of the prostate. [Cancer Res 2007;67(24):11970–9]

AbstractRelatively little attention has been paid to the role of virotherapy in promoting antitumor immune responses. Here, we show that CD8+ T cells are critical for the efficacy of intratumoral vesicular stomatitis virus virotherapy and are induced against both virally encoded and tumor-associated immunodominant epitopes. We tested three separate immune interventions to increase the frequency/activity of activated antitumoral T cells. Depletion of Treg had a negative therapeutic effect because it relieved suppression of the antiviral immune response, leading to early viral clearance. In contrast, increasing the circulating levels of tumor antigen–specific T cells using adoptive T cell transfer therapy, in combination with intratumoral virotherapy, generated significantly improved therapy over either adoptive therapy or virotherapy alone. Moreover, the incorporation of a tumor-associated antigen within the oncolytic vesicular stomatitis virus increased the levels of activation of naïve T cells against the antigen, which translated into increased antitumor therapy. Therefore, our results show that strategies which enhance immune activation against tumor-associated antigens can also be used to enhance the efficacy of virotherapy. [Cancer Res 2007;67(6):2840–7]

AbstractAdoptive T-cell transfer has achieved significant clinical success in advanced melanoma. However, therapeutic efficacy is limited by poor T-cell survival after adoptive transfer and by inefficient trafficking to tumor sites. Here, we report that intratumoral expression of the chemokine CCL21 enhances the efficacy of adoptive T-cell therapy in a mouse model of melanoma. Based on our novel observation that CCL21 is highly chemotactic for activated OT-1 T cells in vitro and down-regulates expression of CD62L, we hypothesized that tumor cell–mediated expression of this chemokine might recruit, and retain, adoptively transferred T cells to the sites of tumor growth. Mice bearing metastatic tumors stably transduced with CCL21 survived significantly longer following adoptive T-cell transfer than mice bearing non–CCL21-expressing tumors. However, although we could not detect increased trafficking of the adoptively transferred T cells to tumors, tumor-expressed CCL21 promoted the survival and cytotoxic activity of the adoptively transferred T cells and led to the priming of antitumor immunity following T-cell transfer. To translate these observations into a protocol of real clinical usefulness, we showed that adsorption of a retrovirus encoding CCL21 to OT-1 T cells before adoptive transfer increased the therapeutic efficacy of a subsequently administered dose of OT-1 T cells, resulting in cure of metastatic disease and the generation of immunologic memory in the majority of treated mice. These studies indicate a promising role for CCL21 in enhancing the therapeutic efficacy of adoptive T-cell therapy. [Cancer Res 2007;67(1):300–8]