Ultralow-dose binary oncolytic/helper-dependent adenovirus promotes antitumor activity in preclinical and clinical studies.

We show that a binary oncolytic/helper-dependent adenovirus (CAdVEC) that both lyses tumor cells and locally expresses the proinflammatory cytokine IL-12 and PD-L1 blocking antibody has potent antitumor activity in humanized mouse models. On the basis of these preclinical studies, we treated four patients with a single intratumoral injection of an ultralow dose of CAdVEC (NCT03740256), representing a dose of oncolytic adenovirus more than 100-fold lower than used in previous trials. While CAdVEC caused no significant toxicities, it repolarized the tumor microenvironment with increased infiltration of CD8 T cells. A single administration of CAdVEC was associated with both locoregional and abscopal effects on metastases and, in combination with systemic administration of immune checkpoint antibodies, induced sustained antitumor responses, including one complete and two partial responses. Hence, in both preclinical and clinical studies, CAdVEC is safe and even at extremely low doses is sufficiently potent to induce significant tumor control through oncolysis and immune repolarization.

HydrAd: A Helper-Dependent Adenovirus Targeting Multiple Immune Pathways for Cancer Immunotherapy.

For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.

Current development in adenoviral vectors for cancer immunotherapy.

Adenoviruses are well characterized and thus easily modified to generate oncolytic vectors that directly lyse tumor cells and can be "armed" with transgenes to promote lysis, antigen presentation, and immunostimulation. Oncolytic adenoviruses (OAds) are safe, versatile, and potent immunostimulants in patients. Since transgene expression is restricted to the tumor, adenoviral transgenes overcome the toxicities and short half-life of systemically administered cytokines, immune checkpoint blockade molecules, and bispecific T cell engagers. While OAds expressing immunostimulatory molecules ("armed" OAds) have demonstrated anti-tumor potential in preclinical solid tumor models, the efficacy has not translated into significant clinical outcomes as a monotherapy. However, OAds synergize with established standards of care and novel immunotherapeutic agents, providing a multifaceted means to address complexities associated with solid tumors. Critically, armed OAds revitalize endogenous and adoptively transferred immune cells while simultaneously enhancing their anti-tumor function. To properly evaluate these novel vectors and reduce the gap in the cycle between bench-to-bedside and back, improving model systems must be a priority. The future of OAds will involve a multidimensional approach that provides immunostimulatory molecules, immune checkpoint blockade, and/or immune engagers in concert with endogenous and exogenous immune cells to initiate durable and comprehensive anti-tumor responses.

Oncolytic adeno-immunotherapy modulates the immune system enabling CAR T-cells to cure pancreatic tumors.

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients.

Clinical CAR-T Cell and Oncolytic Virotherapy for Cancer Treatment.

Immunotherapy has recently garnered success with the induction of clinical responses in tumors, which are traditionally associated with poor outcomes. Chimeric antigen receptor T (CAR-T) cells and oncolytic viruses (OVs) have emerged as promising cancer immunotherapy agents. Herein, we provide an overview of the current clinical status of CAR-T cell and OV therapies. While preclinical studies have demonstrated curative potential, the benefit of CAR-T cells and OVs as single-agent treatments remains limited to a subset of patients. Combinations of different targeted therapies may be required to achieve efficient, durable responses against heterogeneous tumors, as well as the microenvironment. Using a combinatorial approach to take advantage of the unique features of CAR-T cells and OVs with other treatments can produce additive therapeutic effects. This review also discusses ongoing clinical evaluations of these combination strategies for improved outcomes in treatment of resistant malignancies.

Modeling the Efficacy of Oncolytic Adenoviruses In Vitro and In Vivo: Current and Future Perspectives.

Oncolytic adenoviruses (OAd) selectively target and lyse tumor cells and enhance anti- tumor immune responses. OAds have been used as promising cancer gene therapies for many years and there are a multitude of encouraging pre-clinical studies. However, translating OAd therapies to the clinic has had limited success, in part due to the lack of realistic pre-clinical models to rigorously test the efficacy of OAds. Solid tumors have a heterogenous and hostile microenvironment that provides many barriers to OAd treatment, including structural and immunosuppressive components that cannot be modeled in two-dimensional tissue culture. To replicate these characteristics and bridge the gap between pre-clinical and clinical success, studies must test OAd therapy in three-dimensional culture and animal models. This review focuses on current methods to test OAd efficacy in vitro and in vivo and the development of new model systems to test both oncolysis and immune stimulatory components of oncolytic adenovirotherapy.

Oncolytic Adenovirus Armed with BiTE, Cytokine, and Checkpoint Inhibitor Enables CAR T Cells to Control the Growth of Heterogeneous Tumors.

No single cancer immunotherapy will likely defeat all evasion mechanisms of solid tumors, including plasticity of tumor antigen expression and active immune suppression by the tumor environment. In this study, we increase the breadth, potency, and duration of anti-tumor activity of chimeric antigen receptor (CAR) T cells using an oncolytic virus (OV) that produces cytokine, checkpoint blockade, and a bispecific tumor-targeted T cell engager (BiTE) molecule. First, we constructed a BiTE molecule specific for CD44 variant 6 (CD44v6), since CD44v6 is widely expressed on tumor but not normal tissue, and a CD44v6 antibody has been safely administered to cancer patients. We then incorporated this BiTE sequence into an oncolytic-helper binary adenovirus (CAdDuo) encoding an immunostimulatory cytokine (interleukin [IL]-12) and an immune checkpoint blocker (PD-L1Ab) to form CAdTrio. CD44v6 BiTE from CAdTrio enabled HER2-specific CAR T cells to kill multiple CD44v6+ cancer cell lines and to produce more rapid and sustained disease control of orthotopic HER2+ and HER2-/- CD44v6+ tumors than any component alone. Thus, the combination of CAdTrio with HER2.CAR T cells ensures dual targeting of two tumor antigens by engagement of distinct classes of receptor (CAR and native T cell receptor [TCR]), and significantly improves tumor control and survival.

Oncolytic Viruses Partner With T-Cell Therapy for Solid Tumor Treatment.

Adoptive T-cell immunotherapies, including chimeric antigen receptor-modified T-cells (CAR-T cells), have revolutionized cancer treatment, especially for hematologic malignancies. Clinical success of CAR-T cell monotherapy in solid tumors however, has been only modest. Oncolytic viruses provide direct cancer cell lysis, stimulate systemic immune responses, and have the capacity to provide therapeutic transgenes. Oncolytic virotherapy has shown great promise in many preclinical solid tumor models and the first oncolytic virus has been approved by the FDA for the treatment of advanced melanoma. As monotherapies for solid tumors, oncolytic virotherapy provides only moderate anti-tumor effects. However, due to their complementary modes of action, oncolytic virus and T-cell therapies can be combined to overcome the inherent limitations of each agent. This review focuses on the aspects of oncolytic viruses that enable them to synergize with adoptive T-cell immunotherapies to enhance anti-tumor effects for solid tumors.

Adenovirotherapy Delivering Cytokine and Checkpoint Inhibitor Augments CAR T Cells against Metastatic Head and Neck Cancer.

In solid tumors, chimeric antigen receptor (CAR)-modified T cells must overcome the challenges of the immunosuppressive tumor microenvironment. We hypothesized that pre-treating tumors with our binary oncolytic adenovirus (CAd), which produces local oncolysis and expresses immunostimulatory molecules, would enhance the antitumor activity of HER2-specific CAR T cells, which alone are insufficient to cure solid tumors. We tested multiple cytokines in conjunction with PD-L1-blocking antibody and found that Ad-derived IL-12p70 prevents the loss of HER2.CAR-expressing T cells at the tumor site. Accordingly, we created a construct encoding the PD-L1-blocking antibody and IL-12p70 (CAd12_PDL1). In head and neck squamous cell carcinoma (HNSCC) xenograft models, combining local treatment with CAd12_PDL1 and systemic HER2.CAR T cell infusion improved survival to >100 days compared with approximately 25 days with either approach alone. This combination also controlled both primary and metastasized tumors in an orthotopic model of HNSCC. Overall, our data show that CAd12_PDL1 augments the anti-tumor effects of HER2.CAR T cells, thus controlling the growth of both primary and metastasized tumors.

Armed Oncolytic Adenovirus-Expressing PD-L1 Mini-Body Enhances Antitumor Effects of Chimeric Antigen Receptor T Cells in Solid Tumors.

Chimeric antigen receptor-modified T cells (CAR T cells) produce proinflammatory cytokines that increase expression of T-cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the proinflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body; HDPDL1) as a strategy to enhance CAR T-cell killing. Coadministration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no antitumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T cells enhanced antitumor activity compared with treatment with either HER2.CAR T cells alone or HER2.CAR T cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2+ prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors. Cancer Res; 77(8); 2040-51. ©2017 AACR.

Recent advances in oncolytic adenovirus therapies for cancer.

Oncolytic adenoviruses (Onc.Ads) selectively replicate in and lyse cancer cells and are therefore commonly used vectors in clinical trials for cancer gene therapy. Building upon the well-characterized adenoviral natural tropism, genetic modification of Onc.Ad can enhance/regulate their transduction and replication within specific cancer cell types. However, Onc.Ad-mediated tumor cell lysis cannot fully eliminate tumors. The hostile tumor microenvironment provides many barriers to efficient oncolytic virotherapy, as tumors develop structure and immune-evasion mechanisms in order to grow and ultimately spread. For these reasons, Onc.Ads modified to deliver structural or immune modulatory molecules (Armed Onc.Ads) have been developed to overcome the physical and immunological barriers of solid tumors. The combination of oncolysis with tumor microenvironment modulation/destruction may provide a promising platform for Ad-based cancer gene therapy.