Tooba A. Cheema

Multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model

Glioblastoma (World Health Organization grade IV) is an aggressive adult brain tumor that is inevitably fatal despite surgery, radiation, and chemotherapy. Treatment failures are attributed to combinations of cellular heterogeneity, including a subpopulation of often-resistant cancer stem cells, aberrant vasculature, and noteworthy immune suppression. Current preclinical models and treatment strategies do not incorporate or address all these features satisfactorily. Herein, we describe a murine glioblastoma stem cell (GSC) model that recapitulates tumor heterogeneity, invasiveness, vascularity, and immunosuppressive microenvironment in syngeneic immunocompetent mice and should prove useful for a range of therapeutic studies. Using this model, we tested a genetically engineered oncolytic herpes simplex virus that is armed with an immunomodulatory cytokine, interleukin 12 (G47∆-mIL12). G47Δ-mIL12 infects and replicates similarly to its unarmed oncolytic herpes simplex virus counterpart in mouse 005 GSCs in vitro, whereas in vivo, it significantly enhances survival in syngeneic mice bearing intracerebral 005 tumors. Mechanistically, G47∆-mIL12 targets not only GSCs but also increases IFN-γ release, inhibits angiogenesis, and reduces the number of regulatory T cells in the tumor. The increased efficacy is dependent upon T cells, but not natural killer cells. Taken together, our findings demonstrate that G47Δ-mIL12 provides a multifaceted approach to targeting GSCs, tumor microenvironment, and the immune system, with resultant therapeutic benefit in a stringent glioblastoma model.

Enhanced Antitumor Efficacy of Low-Dose Etoposide with Oncolytic Herpes Simplex Virus in Human Glioblastoma Stem Cell Xenografts

Purpose: Glioblastoma (GBM) inevitably recurs despite surgery, radiation, and chemotherapy. A subpopulation of tumor cells, GBM stem cells (GSC), has been implicated in this recurrence. The chemotherapeutic agent etoposide is generally reserved for treating recurrent tumors; however, its effectiveness is limited due to acute and cumulative toxicities to normal tissues. We investigate a novel combinatorial approach of low-dose etoposide with an oncolytic HSV to enhance antitumor activity and limit drug toxicity. Experimental Design: In vitro, human GBM cell lines and GSCs were treated with etoposide alone, oncolytic herpes simplex virus (oHSV) G47Δ alone, or the combination. Cytotoxic interactions were analyzed using the Chou–Talalay method, and changes in caspase-dependent apoptosis and cell cycle were determined. In vivo, the most etoposide-resistant human GSC, BT74, was implanted intracranially and treated with either treatment alone or the combination. Analysis included effects on survival, therapy-associated adverse events, and histologic detection of apoptosis. Results: GSCs varied in their sensitivity to etoposide by over 50-fold in vitro, whereas their sensitivity to G47Δ was similar. Combining G47Δ with low-dose etoposide was moderately synergistic in GSCs and GBM cell lines. This combination did not enhance virus replication, but significantly increased apoptosis. In vivo, the combination of a single cycle of low-dose etoposide with G47Δ significantly extended survival of mice-bearing etoposide–insensitive intracranial human GSC–derived tumors. Conclusions: The combination of low-dose etoposide with G47Δ increases survival of mice-bearing intracranial human GSC–derived tumors without adverse side effects. These results establish this as a promising combination strategy to treat resistant and recurrent GBM. Clin Cancer Res; 17(23); 7383–93. ©2011 AACR.

Oncolytic herpes simplex virus vectors and chemotherapy: are combinatorial strategies more effective for cancer?

Despite aggressive treatments, including chemotherapy and radiotherapy, cancers often recur owing to resistance to conventional therapies. Oncolytic viruses such as oncolytic herpes simplex virus (oHSV) represent an exciting biological approach to cancer therapy. A range of viral mutations has been engineered into HSV to engender oncolytic activity. While oHSV as a single agent has been tested in a number of cancer clinical trials, preclinical studies have demonstrated enhanced efficacy when it is combined with cytotoxic anticancer drugs. Among the strategies that will be discussed in this article are combinations with standard-of-care chemotherapeutics, expression of prodrug-activating enzymes to enhance chemotherapy and small-molecule inhibitors. The combination of oHSV and chemotherapy can achieve much more efficient cancer cell killing than either single agent alone, often through synergistic interactions. This can be clinically important not just for improving efficacy but also for permitting lower and less toxic chemotherapeutic doses. The viral mutations in an oHSV vector often determine the favorability of its interactions with chemotherapy, just as different cancer cells, due to genetic alterations, vary in their response to chemotherapy. As chemotherapeutics are often the standard of care, combining them with an investigational new drug, such as oHSV, is clinically easier than combining multiple novel agents. As has become clear for most cancer therapies, multimodal treatments are usually more effective. In this article, we will discuss the recent progress of these combinatorial strategies between virotherapy and chemotherapy and future directions.

Identification of the ENT1 Antagonists Dipyridamole and Dilazep as Amplifiers of Oncolytic Herpes Simplex Virus-1 Replication

Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity.

Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models

Oncolytic herpes simplex virus (oHSV)-1-based vectors selectively replicate in tumor cells causing direct killing, that is, oncolysis, while sparing normal cells. The oHSVs are promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the interleukin (IL)-12-secreting NV1042 oHSV with microtubule disrupting agents (MDAs) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDAs screened, which displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube-forming assays, VB was found to be highly effective at inhibiting tube formation of human umbilical vein endothelial cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines, and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31+ cells as compared with other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting antitumor and antiangiogenic activities of oHSVs and MDAs.

Immunovirotherapy for the treatment of glioblastoma

We have recently described a new murine model of glioblastoma, generated by the implantation of syngeneic glioblastoma stem cells into immunocompetent mice, that recapitulates the salient histopathological and immunological features of the human disease. We employed this model to demonstrate the multifaceted activity of an oncolytic herpes simplex virus genetically modified to express interleukin-12, G47∆-IL12.

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell–deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell–activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.Patients with glioblastoma experience lymphopenia and sequestration of T cells in the bone marrow, which is recapitulated in mice with brain tumors, where the reversible nature of this effect is demonstrated by an approach that enables the efficacy of other immunotherapeutics.

Abstract 4605: Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody

B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody-Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co-stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti-tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93-103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti-BCMA antibody, SEA-BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA-BCMA - opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA-BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi-faceted activity of ACTR T cells in combination with the anti-BCMA antibody, SEA-BCMA, for clinical consideration in multiple myeloma patients. Citation Format: Tooba Cheema, Taylor Hickman, Katie O9Callaghan, Lori Westendorf, Luke Manlove, Shyra Gardai, Allison Nelson, Ryan Boomer, Kathleen McGinness, Birgit Schultes, Seth Ettenberg, Django Sussman, Heather Huet. Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2017-4605

Abstract 5389: Oncolytic herpes simplex virus expressing interleukin-12 for treating glioma stem cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma multiforme (GBM) is an aggressive adult brain tumor that despite surgery, radiation and chemotherapy, inevitably recurs. This tumor recurrence is thought to be due to a subpopulation of cells with stem cell-like properties called glioma stem cells (GSCs), and specific targeting of these GSCs might improve GBM treatment. Oncolytic herpes simplex virus (oHSV) vectors are genetically engineered to selectively replicate in, and kill cancer cells, without harming normal tissue, and have been shown to be safe in glioma clinical trials. Our group has shown that oHSV G47delta (G47D) can kill human GSCs, yet its efficacy in vivo was insufficient. Moreover, it is not well understood if GSCs can be targeted effectively by an anti-tumor immune response, partly due to the lack of immune-competent mouse models of GSCs. In this study, we hypothesize that arming G47D with interleukin-12 (IL-12), a critical cytokine involved in adaptive and innate immune responses as well as anti-angiogenesis, will be more effective at targeting GSCs, and tested this using a new syngeneic mouse model of GSCs. We characterized mouse 005 GSCs (obtained from I. Verma, UCSD) which constitutively express H-Ras and are p53-/+. They exhibit stem cells markers such as nestin and CD133, could be differentiated into neuronal and glial phenotype, and could form tumors in C57BL/6 mice with characteristic GBM necrosis, giant cells and CD31-positive vasculature. These 005 mGSCs expressed MHC/NK ligand markers, but lacked significant expression of co-stimulatory signaling molecules for T cells as observed by flow cytometry. In vitro, treatment of 005 GSCs with increasing concentrations of G47D-Empty (−E; without transgene) or G47D-IL12 resulted in increase in cytotoxicity with similar EC50 values (MOI∼0.1 at 4 days). G47D-IL12 also replicated well in 005 mGSCs leading to a significant release of IL-12 as measured by ELISA. Intracranial tumors established by implanting 005 mGSC in C57BL/6 mice were then treated with two intratumoral injections of either G47D-E or G47D-IL12 oHSV. This resulted in a significant inhibition in tumor growth with extension of survival with G47D-E (median survival 40.5 days; p< 0.03) and G47D-IL12 (median survival 56 days; p< 0.0001) compared with control saline-injected mice with a median survival of 37 days. Importantly, there was a significant increase in survival of mice treated with G47D-IL12 (p< 0.0003) when compared with G47D-E. This increase in survival, potentially due to increased immune response and/or decrease in angiogenesis, is currently under investigation. This is the first demonstration that GSC can be effectively targeted with oHSV-IL12 in a syngeneic GSC mouse model, showing its marked efficacy over G47D-E. This may be a promising strategy to eradicate glioma cell populations through direct oncolysis combined with enhancing anti-tumor immunity as well as altering the tumor vasculature microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2011-5389