Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

Texas Tech University Health Sciences Center

Single-agent immunotherapy, including with immune checkpoint inhibition with anti-PD-1 antibody, has not extended survival in patients with malignant glioma. However, PD-1 inhibition may still play a role in combination immunotherapy with multiple agents. In this study, we evaluated anti-PD-1 antibody treatment in combination with multiple approaches, including vaccination and agonist anti-OX40 immunotherapy, as well as triple combination immunotherapy with each of the above agents in a murine glioma model. Treatments were delivered on days 3,6, and 9 after intracranial implantation of glioma cells in the right frontal lobes of the mice. Vaccination consisted of subcutaneous implantation of irradiated GL261 cells engineered to express GM-CSF. We harvested splenocytes and brain tissue 18 days after glioma implantation and analyzed them by ELISPOT and flow cytometry, respectively. Treated mice surviving for 120 days were challenged with implantation of large numbers of GL261 cells and either followed for survival or sacrificed for study of the memory response. Survival was assessed by the Kaplan-Meier method and the log-rank test. Means were compared by the 2-tailed student's t-test. We report that combining anti-PD-1 immunotherapy with either vaccination or agonist anti-OX40 immunotherapy improves survival in GL261-bearing mice compared with any of the above as monotherapy. Triple combination immunotherapy with vaccination, anti-PD-1 antibody, and agonist anti-OX40 antibody results in long-term survival in all mice. Triple combination immunotherapy resulted in an elevated CD4+/CD8 + T lymphocyte ratio amongst tumor-infiltrating lymphocytes as well as a diminished fraction of regulatory T lymphocytes, likely reflective of a more vigorous Th1 antitumor response.

Triple combination immunotherapy with GVAX, anti-PD-1 monoclonal antibody, and agonist anti-OX40 monoclonal antibody is highly effective against murine intracranial glioma.

Multi-color flow cytometry is a standard laboratory protocol, which is regularly used to analyze tumor-infiltrating immune cell subsets. Oncolytic herpes simplex virus has shown promise in treating various types of cancers, including deadly glioblastoma. Intracranial/intratumoral treatment with oncolytic herpes simplex virus expressing interleukin 12, ., immunovirotherapy results in induction of anti-tumor immune responses and tumor infiltration of a variety of immune cells. Multi-color flow cytometry is employed to characterize immune cells in the tumor microenvironment. Here, we describe a step-by-step 11-color flow cytometry protocol to stain tumor-infiltrating immune cells in glioblastoma following oncolytic herpes virotherapy. We also describe a method to identify HSV-1 glycoprotein-B-specific CD8 T cells using fluorochrome-conjugated major histocompatibility complex multimers. The multimers carry major histocompatibility peptide complexes, which have the ability to interact and bind to T cell receptors present on the surface of T cells; allowing identification of T cells (., CD8) reactive to a desired antigen. This multimer staining can be used in conjunction with the multi-parametric flow cytometry staining. Brain tumor quadrants are harvested, minced, enzymatically digested, immune cells are isolated by positive selection, single cells are counted and blocked for Fc receptors, cells are incubated with dye and/or color-conjugated antibodies, and flow cytrometry is performed using a BD LSRII flow cytometer. The protocol described herein is also applicable to stain immune cells in other mouse and human tumors or in any desired tissues.

Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma.

Immunohistochemistry (IHC) is an integral laboratory staining technique, which is used for the detection of immune cells in mouse/human tissues or tumors. Oncolytic herpes simplex virus (oHSV) treatment or virotherapy of solid tumors results in antitumor immune responses and infiltration of a variety of immune cells into the tumor. Here, we describe a step-by-step chromogen/substrate-based single- and dual-color IHC protocol to stain immune cells in formalin-fixed, paraffin-embedded mouse glioblastoma (GBM) brain tumor sections after oHSV virotherapy. Tumor sections are deparaffinized with xylene, then gradually rehydrated using ethanol, followed by heat-mediated antigen retrieval using appropriate buffers. Tumor sections are incubated with primary antibodies, which detect a specific immune cell antigen, then incubated with peroxidase- or phosphatase-labeled secondary antibodies, followed by incubation with a color-producing substrate and color visualization (of immune cells) by light microscopy. The protocol described herein is also applicable to detect immune cells in other mouse and human tumors or organs after other forms of immunotherapy.

Immunohistochemistry for Tumor-Infiltrating Immune Cells After Oncolytic Virotherapy.

Oncolytic viruses (OVs) are genetically modified or naturally occurring viruses, which preferentially replicate in and kill cancer cells while sparing healthy cells, and induce anti-tumor immunity. OV-induced tumor immunity can be enhanced through viral expression of anti-tumor cytokines such as interleukin 12 (IL-12). IL-12 is a potent anti-cancer agent that promotes T-helper 1 (Th1) differentiation, facilitates T-cell-mediated killing of cancer cells, and inhibits tumor angiogenesis. Despite success in preclinical models, systemic IL-12 therapy is associated with significant toxicity in humans. Therefore, to utilize the therapeutic potential of IL-12 in OV-based cancer therapy, 25 different IL-12 expressing OVs (OV-IL12s) have been genetically engineered for local IL-12 production and tested preclinically in various cancer models. Among OV-IL12s, oncolytic herpes simplex virus encoding IL-12 (OHSV-IL12) is the furthest along in the clinic. IL-12 expression locally in the tumors avoids systemic toxicity while inducing an efficient anti-tumor immunity and synergizes with anti-angiogenic drugs or immunomodulators without compromising safety. Despite the rapidly rising interest, there are no current reviews on OV-IL12s that exploit their potential efficacy and safety to translate into human subjects. In this article, we will discuss safety, tumor-specificity, and anti-tumor immune/anti-angiogenic effects of OHSV-IL12 as mono- and combination-therapies. In addition to OHSV-IL12 viruses, we will also review other IL-12-expressing OVs and their application in cancer therapy.

Oncolytic Virus Encoding a Master Pro-Inflammatory Cytokine Interleukin 12 in Cancer Immunotherapy.

Triple-negative breast cancer (TNBC) is a difficult-to-treat disease with high rates of local recurrence, distant metastasis, and poor overall survival with existing therapies. Thus, there is an unmet medical need to develop new treatment regimen(s) for TNBC patients. An oncolytic herpes simplex virus encoding a master anti-tumor cytokine, interleukin 12, (designated G47Δ-mIL12) selectively kills cancer cells while inducing anti-tumor immunity. G47Δ-mIL12 efficiently infected and killed murine (4T1 and EMT6) and human (HCC1806 and MDA-MB-468) mammary tumor cells .  in the 4T1 syngeneic TNBC model, it significantly reduced primary tumor burden and metastasis, both at early and late stages of tumor development. The virus-induced local and abscopal effects were confirmed by significantly increased infiltration of CD45 leukocytes and CD8 T cells, and reduction of granulocytic and monocytic MDSCs in tumors, both treated and untreated contralateral, and in the spleen. Significant trafficking of dendritic cells (DCs) were only observed in spleens of virus-treatment group, indicating that DCs are primed and activated in the tumor-microenvironment following virotherapy, and trafficked to lymphoid organs for activation of immune cells, such as CD8 T cells. DC priming/activation could be associated with virally enhanced expression of several antigen processing/presentation genes in the tumor microenvironment, as confirmed by NanoString gene expression analysis. Besides DC activation/priming, G47Δ-mIL12 treatment led to up-regulation of CD8 T cell activation markers in the tumor microenvironment and inhibition of tumor angiogenesis. The anti-tumor effects of G47Δ-mIL12 treatment were CD8-dependent. These studies illustrate the ability of G47Δ-mIL12 to immunotherapeutically treat TNBC.

Oncolytic Herpes Simplex Virus Encoding IL12 Controls Triple-Negative Breast Cancer Growth and Metastasis.

Temozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system, both beneficially and negatively, understanding how TMZ interacts with immunotherapeutics is important. Oncolytic herpes simplex virus (oHSV) is a new class of cancer therapeutic with both cytotoxic and immunostimulatory activities. Here, we examine the combination of TMZ and an oHSV encoding murine interleukin 12, G47Δ-mIL12, in a mouse immunocompetent GBM model generated from non-immunogenic 005 GBM stem-like cells (GSCs.

Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma.

Melanoma continues to be an aggressive and deadly form of skin cancer while therapeutic options are continuously developing in an effort to provide long-term solutions for patients. Immunotherapeutic strategies incorporating antibody-drug conjugates (ADCs) have seen varied levels of success across tumor types and represent a promising approach for melanoma. This review will explore the successes of FDA-approved ADCs to date compared to the ongoing efforts of melanoma-targeting ADCs. The challenges and opportunities for future therapeutic development are also examined to distinguish how ADCs may better impact individuals with malignancies such as melanoma.

Antibody-drug conjugates: an evolving approach for melanoma treatment.

Glioblastoma (GBM) is a lethal primary malignant brain tumor with no current effective treatments. The recent emergence of immuno-virotherapy and FDA approval of T-VEC have generated a great expectation towards oncolytic herpes simplex viruses (oHSVs) as a promising treatment option for GBM. Since the generation and testing of the first genetically engineered oHSV in glioma in the early 1990s, oHSV-based therapies have shown a long way of great progress in terms of anti-GBM efficacy and safety, both preclinically and clinically. Here, we revisit the literature to understand the recent advancement of oHSV in the treatment of GBM. In addition, we discuss current obstacles to oHSV-based therapies and possible strategies to overcome these pitfalls.

Dipongkor Saha

Department of Immunotherapeutics and Biotechnology,
Jerry H. Hodge School of Pharmacy,
Department of Immunotherapeutics and Biotechnology, Jerry H. Hodge School of Pharmacy

Texas Tech University Health Sciences Center

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Dipongkor Saha

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Immunotherapeutics and Biotechnology,Jerry H. Hodge School of Pharmacy,Department of Immunotherapeutics and Biotechnology, Jerry H. Hodge School of Pharmacy

Texas Tech University Health Sciences Center

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Department of Immunotherapeutics and Biotechnology,
Jerry H. Hodge School of Pharmacy,
Department of Immunotherapeutics and Biotechnology, Jerry H. Hodge School of Pharmacy