Magesh Sathaiah

Oncolytic viruses as therapeutic cancer vaccines

Oncolytic viruses (OVs) are tumor-selective, multi-mechanistic antitumor agents. They kill infected cancer and associated endothelial cells via direct oncolysis, and uninfected cells via tumor vasculature targeting and bystander effect. Multimodal immunogenic cell death (ICD) together with autophagy often induced by OVs not only presents potent danger signals to dendritic cells but also efficiently cross-present tumor-associated antigens from cancer cells to dendritic cells to T cells to induce adaptive antitumor immunity. With this favorable immune backdrop, genetic engineering of OVs and rational combinations further potentiate OVs as cancer vaccines. OVs armed with GM-CSF (such as T-VEC and Pexa-Vec) or other immunostimulatory genes, induce potent anti-tumor immunity in both animal models and human patients. Combination with other immunotherapy regimens improve overall therapeutic efficacy. Coadministration with a HDAC inhibitor inhibits innate immunity transiently to promote infection and spread of OVs, and significantly enhances anti-tumor immunity and improves the therapeutic index. Local administration or OV mediated-expression of ligands for Toll-like receptors can rescue the function of tumor-infiltrating CD8+ T cells inhibited by the immunosuppressive tumor microenvironment and thus enhances the antitumor effect. Combination with cyclophosphamide further induces ICD, depletes Treg, and thus potentiates antitumor immunity. In summary, OVs properly armed or in rational combinations are potent therapeutic cancer vaccines.

C-stage in Colon Cancer: Implications of Carcinoembryonic Antigen Biomarker in Staging, Prognosis, and Management

BACKGROUND The American Joint Committee on Cancer (AJCC) has proposed the inclusion of pretreatment serum carcinoembryonic antigen (CEA) level (C-stage) into the conventional TNM staging system of colon cancer. We assessed the prognosis of various stages of colon cancer after such an inclusion. METHODS Data for all patients (N = 17 910) diagnosed with colonic adenocarcinoma (AJCC stages I, IIA, IIB, IIC, IIIA, IIIB, IIIC, and IV, based on TNM staging system) between January 1, 2004, and December 31, 2004, with a median follow-up of 27 months (range 0-35 months), were collected from the Surveillance, Epidemiology, and End Results database. C-stage (C0-stage = normal CEA level; C1-stage = elevated CEA level) was assigned to all patients with available CEA information (n = 9083). Multivariable analyses using Cox proportional hazards models were used to identify independent factors associated with prognosis. Prognosis of overall stages (AJCC stages I-IV and C0 or C1) was analyzed using Kaplan-Meier survival curves. All statistical tests were two-sided. RESULTS C1-stage was independently associated with a 60% increased risk of overall mortality (hazard ratio of death = 1.60, 95% confidence interval = 1.46 to 1.76, P < .001). Overall survival was decreased in patients with C1-stage cancer compared with C0-stage cancer of the respective overall stages (P < .05). Similarly, decreased overall survival was noted in patients with stage I C1 cancer compared with stage IIA C0 or stage IIIA C0 cancer (P < .001), in patients with stage IIA C1 cancer compared with stage IIIA C0 (P < .001), and in patients with stage IIB C1 or stage IIC C1 cancer compared with stage IIIB C0 cancer (P < .001). CONCLUSIONS C-stage was an independent prognostic factor for colon cancer. The results support routine preoperative CEA testing and C-staging upon diagnosis of colon cancer and the inclusion of C-stage in the conventional TNM staging of colon cancer.

The combination of immunosuppression and carrier cells significantly enhances the efficacy of oncolytic poxvirus in the pre-immunized host

Pre-existing antipoxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic double-deleted vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B-cell knockout mice, we confirmed that the neutralizing antibodies had a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (interleukin (IL)-10hi/IL-12low) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results showed the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy.

Modulation of chemokines in the tumor microenvironment enhances oncolytic virotherapy for colorectal cancer

An oncolytic poxvirus such as vvDD-CXCL11 can generate potent systemic antitumor immunity as well as targeted oncolysis, yet the antitumor effect is limited probably due to limited homing to and suppressed activity of tumor-specific adaptive immune cells in the tumor microenvironment (TME). We reasoned that a chemokine modulating (CKM) drug cocktail, consisting of IFN-α, poly I:C, and a COX-2 inhibitor, may skew the chemokine (CK) and cytokine profile into a favorable one in the TME, and this pharmaceutical modulation would enhance both the trafficking into and function of antitumor immune cells in the TME, thus increasing therapeutic efficacy of the oncolytic virus. In this study we show for the first time in vivo that the CKM modulates the CK microenvironment but it does not modulate antitumor immunity by itself in a MC38 colon cancer model. Sequential treatment with the virus and then CKM results in the upregulation of Th1-attracting CKs and reduction of Treg-attracting CKs (CCL22 and CXCL12), concurrent with enhanced trafficking of tumor-specific CD8+ T cells and NK cells into the TME, thus resulting in the most significant antitumor activity and long term survival of tumor-bearing mice. This novel combined regimen, with the oncolytic virus (vvDD-CXCL11) inducing direct oncolysis and eliciting potent antitumor immunity, and the CKM inducing a favorable chemokine profile in the TME that promotes the trafficking and function of antitumor Tc1/Th1 and NK cells, may have great utility for oncolytic immunotherapy for cancer.

A rationally designed A34R mutant oncolytic poxvirus: improved efficacy in peritoneal carcinomatosis.

Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV).

Rapid Generation of Multiple Loci-Engineered Marker-free Poxvirus and Characterization of a Clinical-Grade Oncolytic Vaccinia Virus

Recombinant poxviruses, utilized as vaccine vectors and oncolytic viruses, often require manipulation at multiple genetic loci in the viral genome. It is essential for viral vectors to possess no adventitious mutations and no (antibiotic) selection marker in the final product for human patients in order to comply with the guidance from the regulatory agencies. Rintoul et al. have previously developed a selectable and excisable marker (SEM) system for the rapid generation of recombinant vaccinia virus. In the current study, we describe an improved methodology for rapid creation and selection of recombinant poxviruses with multiple genetic manipulations solely based on expression of a fluorescent protein and with no requirement for drug selection that can lead to cellular stress and the risk of adventitious mutations throughout the viral genome. Using this improved procedure combined with the SEM system, we have constructed multiple marker-free oncolytic poxviruses expressing different cytokines and other therapeutic genes. The high fidelity of inserted DNA sequences validates the utility of this improved procedure for generation of therapeutic viruses for human patients. We have created an oncolytic poxvirus expressing human chemokine CCL5, designated as vvDD-A34R-hCCL5, with manipulations at two genetic loci in a single virus. Finally, we have produced and purified this virus in clinical grade for its use in a phase I clinical trial and presented data on initial in vitro characterization of the virus.

Abstract 1544: Combined oncolytic virotherapy and immunotherapy for malignant mesothelioma

Introduction. Oncolytic viruses have therapeutic potential in cancer by selectively replicating within malignant cells and/or other cells in the tumor tissues, causing cell death. Vaccinia virus (VACV) with deletion of viral genes encoding for thymidine kinase and vaccinia growth factor and armed with a chemokine gene encoding CCL5 (vvDD-CCL5) has been previously shown to be safe and efficacious in a murine colon cancer model (Li J. et al., Mol Ther., 19: 650-7; 2011). Here we explored combination therapy with the use of this armed oncolytic virus and immunotherapy in a murine malignant mesothelioma model in syngeneic BALB/c mice. Methods. The viral replication and oncolysis was examined in murine AB12 mesothelioma cancer cells in vitro, and the combination of oncolytic virus, IL-2 and anti-CD25 and anti-CTLA4 antibodies, was investigated in vivo to treat a murine model of peritoneal malignant mesothelioma (PMM) derived from AB12 cells in BALB/c mice. Results. In vitro, vvCCL5 and its parental virus vvDD replicate well in AB12 cancer cells and lead to efficient cytotoxicity in these cancer cells. In vivo biodistribution analysis indicated that both viruses displayed highly tumor selective replication; however, vvDD-CCL5 replicated to a greater extent and displayed increased persistence in the tumor tissue. As a result, vvDD-CCL5 was more efficient than vvDD, working in a viral dose-dependent manner and leading to increased survival of tumor-bearing mice. Combination therapy of virus with antibodies against CD25 and CTLA4 and IL-2 was investigated. Clearly, this has been the best strategy to treat AB12 PMM, achieving the best long-term survival of mice. We are currently investigating its cellular and molecular mechanisms by this combination leading to tumor regression and long-term survival of mice. Conclusions: The chemokine gene-armed oncolytic virus (vvDD-CCL5) is effective in a PMM tumor model. The combination of this virus with an immunologic regimen of Treg depletion and IL-2 treatment has been demonstrated to be highly efficacious for PMM. Further understanding of its cellular and molecular mechanisms of this treatment strategy will facilitate the optimization of combined therapeutic regimens for PMM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1544. doi:1538-7445.AM2012-1544

Abstract 1498: The combination of immunosuppression and carrier cells significantly enhances poxvirus delivery and the efficacy of its oncolytic virotherapy in the pre-immunized host

The pre-existing anti-poxvirus immunity in cancer patients presents a hurdle to successful poxvirus-mediated oncolytic virotherapy. To overcome this hurdle, we have explored strategies of immunosuppression (IS) or/and immune evasion (IE) for successful delivery of an oncolytic vaccinia virus to tumor models in mice pre-vaccinated with a poxvirus. These include transient IS using a cocktail of three immunosuppressive drugs (IS drugs) (tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate) that have been used in organ transplant, carrier cells as a means of IE, or the combined strategy. The three drug cocktail, which targeted multiple components of innate and adaptive immunity, displayed no enhancing effects on viral infection in tumor tissue in the pre-immunized host. Using B cell knockout mice, we confirmed that B cells and the neutralizing antibodies, the later of which were not targeted by the drug cocktail, played a significant role in preventing poxvirus infection. Cancer cells as carrier cells alone were not effective to deliver the virus to tumor. Instead, the application of both IS drugs and carrier cells worked synergistically to promote the delivery to, viral infection, replication and spread inside the tumor mass and thus enhanced the efficacy of oncolytic virotherapy in an ovarian carcinomatosis model. The administraton of these IS drugs led to facilitated development of tumor-associated macrophages (TAMs) into immunosuppressive M2 phenotype (IL-10 hi /IL-12 low ) in the tumor microenvironment. These results revealed a novel mechanism for induction of the immunosuppressive tumor microenvironment by the IS drugs, and demonstrated in principle that it is feasible to treat pre-vaccinated cancer patients with an oncolytic poxvirus when combined with both IS and IE strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1498.