Roshni Ravindranathan

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.

Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

Both anti-PD1/PD-L1 therapy and oncolytic virotherapy have demonstrated promise, yet have exhibited efficacy in only a small fraction of cancer patients. Here we hypothesized that an oncolytic poxvirus would attract T cells into the tumour, and induce PD-L1 expression in cancer and immune cells, leading to more susceptible targets for anti-PD-L1 immunotherapy. Our results demonstrate in colon and ovarian cancer models that an oncolytic vaccinia virus attracts effector T cells and induces PD-L1 expression on both cancer and immune cells in the tumour. The dual therapy reduces PD-L1+ cells and facilitates non-redundant tumour infiltration of effector CD8+, CD4+ T cells, with increased IFN-γ, ICOS, granzyme B and perforin expression. Furthermore, the treatment reduces the virus-induced PD-L1+ DC, MDSC, TAM and Treg, as well as co-inhibitory molecules-double-positive, severely exhausted PD-1+CD8+ T cells, leading to reduced tumour burden and improved survival. This combinatorial therapy may be applicable to a much wider population of cancer patients.

CXCL11-Armed oncolytic poxvirus elicits potent antitumor immunity and shows enhanced therapeutic efficacy

ABSTRACT We have armed a tumor-selective oncolytic vaccinia virus (vvDD) with the chemokine (CK) CXCL11, in order to enhance its ability to attract CXCR3+ antitumor CTLs and possibly NK cells to the tumor microenvironment (TME) and improve its therapeutic efficacy. As expected, vvDD-CXCL11 attracted high numbers of tumor-specific T cells to the TME in a murine AB12 mesothelioma model. Intratumoral virus-directed CXCL11 expression enhanced local numbers of CD8+ CTLs and levels of granzyme B, while reducing expression of several suppressive molecules, TGF-β, COX2, and CCL22 in the TME. Unexpectedly, we observed that vvDD-CXCL11, but not parental vvDD, induced a systemic increase in tumor-specific IFNγ-producing CD8+ T cells in the spleen and other lymph organs, indicating the induction of systemic antitumor immunity. This effect was associated with enhanced therapeutic efficacy and a survival benefit in tumor-bearing mice treated with vvDD-CXCL11, mediated by CD8+ T cells and IFNγ, but not CD4+ T cells. These results demonstrate that intratumoral expression of CXCL11, in addition to promoting local trafficking of T cells and to a lesser extent NK cells, has a novel function as a factor eliciting systemic immunity to cancer-associated antigens. Our data provide a rationale for expressing CXCL11 to enhance the therapeutic efficacy of oncolytic viruses (OVs) and cancer vaccines.

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).

Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer

BackgroundComplement is a central part of both the innate and adaptive immune response and its activation has traditionally been considered part of the immunosurveillance response against cancer. Its pro-inflammatory role and its contribution to the development of many illnesses associated with inflammatory states implicate complement in carcinogenesis.MethodsWe evaluated the role of three protein inhibitors of complement—cobra venom factor, humanized cobra venom factor, and recombinant staphylococcus aureus superantigen-like protein 7—in the setting of a transplantable murine colon cancer model. Outcomes were evaluated by monitoring tumor growth, and flow cytometry, ELISPOT, and quantitative real-time PCR were used to determine the impact of complement inhibition on the host immune response.ResultsComplement inhibitors were effective at depleting complement component C3 in tumor bearing mice and this was temporally correlated with a decreased rate of tumor growth during the establishment of tumors. Treatment with cobra venom factor resulted in increased CD8+ T cells as a percentage of tumor-infiltrating cells as well as a reduced immunosuppressive environment evidenced by decreased myeloid derived suppressor cells in splenocytes of treated mice. Complement inhibition resulted in increased expression of the chemoattractive cytokines CCL5, CXCL10, and CXCL11.DiscussionComplement depletion represents a promising mode of immunotherapy in cancer by its ability to impair tumor growth by increasing the host’s effective immune response to tumor and diminishing the immunosuppressive effect created by the tumor microenvironment and ultimately could be utilized as a component of combination immunotherapy.

Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents

This protocol describes how to induce large numbers of tumor-specific cytotoxic T cells (CTLs) in the spleens and lymph nodes of mice receiving dendritic cell (DC) vaccines and how to modulate tumor microenvironments (TMEs) to ensure effective homing of the vaccination-induced CTLs to tumor tissues. We also describe how to evaluate the numbers of tumor-specific CTLs within tumors. The protocol contains detailed information describing how to generate a specialized DC vaccine with augmented ability to induce tumor-specific CTLs. We also describe methods to modulate the production of chemokines in the TME and show how to quantify tumor-specific CTLs in the lymphoid organs and tumor tissues of mice receiving different treatments. The combined experimental procedure, including tumor implantation, DC vaccine generation, chemokine-modulating (CKM) approaches, and the analyses of tumor-specific systemic and intratumoral immunity is performed over 30-40 d. The presented ELISpot-based ex vivo CTL assay takes 6 h to set up and 5 h to develop. In contrast to other methods of evaluating tumor-specific immunity in tumor tissues, our approach allows detection of intratumoral T-cell responses to nonmanipulated weakly immunogenic cancers. This detection method can be performed using basic laboratory skills, and facilitates the development and preclinical evaluation of new immunotherapies.

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.

Superagonist IL-15-Armed Oncolytic Virus Elicits Potent Antitumor Immunity and Therapy That Are Enhanced with PD-1 Blockade

Oncolytic immunotherapy is a promising novel therapeutic for cancer, and further preclinical studies may maximize its therapeutic efficacy. In this study, we construct a novel oncolytic vaccinia virus (VV) expressing a superagoinst IL-15, a fusion protein of IL-15 and IL-15Ralpha. This virus, named vvDD-IL15-Rα, possesses similar replication efficiency as the parental virus vvDD yet leads to significantly more regression of the disease and extends the survival of mice bearing MC38 colon or ID8 ovarian cancer. This novel virus elicits potent adaptive antitumor immunity as shown by ELISPOT assays for interferon-gamma-secreting CD8+ T cells and by the rejection of tumor implants upon re-challenge in the mice, which were previously cured by vvDD-IL15-Rα treatment. In vivo cell depletion assays with antibodies showed that this antitumor activity is highly dependent on CD8+ T cells but much less so on CD4+ T cells and NK cells. Finally, the combination of the oncolytic immunotherapy with anti-PD-1 antibody dramatically improves the therapeutic outcome compared to either anti-PD-1 alone or vvDD-IL15-Rα alone. These results demonstrate that the IL-15-IL-15Rα fusion protein-expressing OV elicits potent antitumor immunity, and rational combination with PD-1 blockade leads to dramatic tumor regression and prolongs the survival of mice bearing colon or ovarian cancers.

Ex-Th17 Foxp3+ T cells - a novel subset of Foxp3+ T cells induced in cancer

Th17 and regulatory T (Treg) cells are integral in maintaining immune homeostasis and Th17-Treg misbalance associates with inflammation. We demonstrate that in addition to natural (n)Treg and induced (i)Treg cells developed from naive precursors, Th17 cells are a novel source of Foxp3+ cells by converting into ex-Th17 Foxp3+ cells, and this helps to reconcile the contradictory information about the relevance in particularly of Th17 subset in immune surveillance. We identified IL-17A+Foxp3+ double-positive and ex-IL-17-producing IL-17A-Foxp3+ T cells to be the underlying mechanism of immune regulation in mesenchymal stem cell-mediated prolonged allograft survival. Further, we identified accumulation of IL17A+Foxp3+ and ex-Th17 Foxp3+ cells in tumor bearing mice, indicating progressive direct Th17-into-Treg cell conversion as a novel phenomenon in cancer. Moreover, we determined the importance of the Th17 cell plasticity for tumor induction and/or progression in ROR-g-/- mice. Our data indicate that RORgt is required not only for Th17 development, but also for effective Treg cell induction. TGF-b1 induced Foxp3 expression was reduced in ROR-g -/- cells. Further, tumor bearing ROR-g-/- mice showed significantly less Foxp3+ Treg cells, but higher IFNg+ Tcells compared to wild type animals. Increased infiltration of IL17+ and FoxP3+ CD4+ T cells in the human ovarian cancer ascites, with the presence of a distinct IL17+FoxP3+ subset, and a significant correlation between tumor-associated Th17 and Treg cells demonstrates the existence of Th17-Foxp3+ T cell inter-relationship in cancer patients. Yin-yang of IL17+ and Foxp3+ is important principle for improved clinical approaches targeting responses against self, allo and/or neo-self.