Díaz, Rosa María

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.

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]

AbstractWe have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.

AbstractMultiple myeloma is a disseminated neoplasm of terminally differentiated plasma cells that is incurable with currently available therapies. Although the disease is radiosensitive, external beam radiation leads to significant toxicity due to sensitive end-organ damage. Thus, genetic approaches for therapy are required. We hypothesized that the incorporation of immunoglobulin promoter and enhancer elements in a self-inactivating (SIN) lentiviral vector should lead to specific and high-level transgene expression in myeloma cells. A SIN lentivector with enhanced green fluorescent protein (EGFP) expression under the control of a minimal immunoglobulin promoter as well as the Kappa light chain intronic and 3′ enhancers transduced myeloma cell lines with high efficiency (30%-90%). EGFP was expressed at a high level in myeloma cells but silent in all nonmyeloma cell lines tested compared with the cytomegalovirus (CMV) promoter/enhancer. Transduction of myeloma cells with the targeted vector coding for the human sodiumiodide symporter (hNIS) led to hNIS expression by these cells allowing them to concentrate radioiodine up to 18-fold compared with controls. Tumor xenografts in severe combined immunodeficiency mice expressing hNIS could be imaged using iodine-123 (123I) and shown to retain iodide for up to 48 hours. These tumor xenografts were completely eradicated by a single dose of the therapeutic isotope iodine-131 (131I) without evidence of recurrence up to 5 months after therapy. We conclude that lentivectors can be transcriptionally targeted for myeloma cells and the use of hNIS as a therapeutic gene for myeloma in combination with 131I needs further exploration.