Melcher, Alan

Background The aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%–60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations. Methods Using a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies. Results We characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy. Conclusions The combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

Abstract COVID-19 patients show significant clinical heterogeneity in presentation and outcomes that makes pandemic control and strategy difficult; optimising management requires a systems biology approach of understanding the disease. Here we sought to understand and infer complex system-wide changes in patients infected with coronaviruses (SARS-CoV and SARS-CoV-2; n=38 and 57 samples) at two different disease stages compared with healthy individuals (n=16) and patients with other infections (n=144). We applied inferential statistics/machine-learning approaches (the COVID-engine platform) to RNA profiles derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, an integrated blood-based gene signatures distinguished acute-like (mimicking coronavirus-infected patients with prolonged hospitalisation) from recovering-like patients. These signatures also hierarchically represented systems-level parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle confirmatory observations included PBMC-associated increases in ACE2, cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage had significantly enhanced TNF, IFN-g, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, PBMC-derived surrogate-based approach revealed overlapping genes associated with comorbidities (associated diabetes), and disease-like symptoms (associated with thromboembolism, pneumonia, lung disease and septicaemia). Overall, our study involving PBMC-based RNA profiling may further help understand complex and variable systems-wide responses displayed by coronavirus-infected patients.

Abstract Purpose: The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novel ICAM-1–targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer. Patients and Methods: Fifteen patients enrolled in this “window of opportunity” phase I study, exposing primary bladder cancers to CAVATAK prior to surgery. The first 9 patients received intravesical administration of monotherapy CAVATAK; in the second stage, 6 patients received CAVATAK with a subtherapeutic dose of mitomycin C, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose (MTD). Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, antitumor activity, and viral-induced changes in resected tissue. Results: Clinical activity of CAVATAK was demonstrated by induction of tumor inflammation and hemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumor in 1 patient. Whether used alone or in combination with mitomycin C, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines, as well as the induction of the innate activator RIG-I, compared with bladder cancer tissue from untreated patients. No significant toxicities were reported in any patient, from either virus or combination therapy. Conclusions: The acceptable safety profile of CAVATAK, proof of viral targeting, replication, and tumor cell death together with the virus-mediated increases in “immunological heat” within the tumor microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.

Abstract Antitumor T-cell responses raised by first-line therapies such as chemotherapy, radiation, tumor cell vaccines, and viroimmunotherapy tend to be weak, both quantitatively (low frequency) and qualitatively (low affinity). We show here that T cells that recognize tumor-associated antigens can directly kill tumor cells if used at high effector-to-target ratios. However, when these tumor-reactive T cells were present at suboptimal ratios, direct T-cell–mediated tumor cell killing was reduced and the ability of tumor cells to evolve away from a coapplied therapy (oncolytic or suicide gene therapy) was promoted. This T-cell–mediated increase in therapeutic resistance was associated with C to T transition mutations that are characteristic of APOBEC3 cytosine deaminase activity and was induced through a TNFα and protein kinase C–dependent pathway. Short hairpin RNA inhibition of endogenous APOBEC3 reduced rates of tumor escape from oncolytic virus or suicide gene therapy to those seen in the absence of antitumor T-cell coculture. Conversely, overexpression of human APOBEC3B in tumor cells enhanced escape from suicide gene therapy and oncolytic virus therapy both in vitro and in vivo. Our data suggest that weak affinity or low frequency T-cell responses against tumor antigens may contribute to the ability of tumor cells to evolve away from first-line therapies. We conclude that immunotherapies need to be optimized as early as possible so that, if they do not kill the tumor completely, they do not promote treatment resistance.

Abstract Tumor cells are able to evade cytotoxic therapies through the accumulation of genomic mutations and rapid evolution. In the case of oncolytic virotherapy, understanding the mechanisms by which cancer cells develop resistance to infection and lysis is critical to the development of more effective viral-based platforms. Here, we identify APOBEC3 as an important factor involved in the restriction of vesicular stomatitis virus (VSV). We show that VSV infection of B16 murine melanoma cells upregulated APOBEC3 in an IFNβ-dependent manner, which was responsible for the evolution of virus-resistanT-cell populations and suggested that APOBEC3 expression promoted the acquisition of a virus-resistant phenotype. ShRNA knockdown of APOBEC3 in B16 cells diminished their capacity to develop resistance to VSV infection in vitro, and enhanced the therapeutic effect of VSV in vivo. Similarly, overexpression of human APOBEC3B promoted the acquisition of resistance to oncolytic VSV in murine melanoma and glioblastoma lines, as well as in a human melanoma cell line in vitro and in vivo. Finally, we demonstrate that progeny virus obtained after passage through APOBEC3B overexpressing cells contained more defective interfering particles, thereby indicating that APOBEC3B directly affected the fitness of VSV, an RNA virus that has not previously been identified to be restricted by APOBEC3B. This research identifies APOBEC3 enzymes as key players to target in order to improve the efficacy of oncolytic viruses as well as broader nucleic acid-based therapeutic platforms. Citation Format: Amanda L. Huff, Phonphimon Wongthida, Timothy Kottke, Jill Thompson, Christopher B. Driscoll, Matthew Schuelke, Kevin G. Shim, Reuben S. Harris, Amy Molan, Jose S. Pulido, Peter J. Selby, Kevin J. Harrington, Alan Melcher, Laura Evgin, Richard Vile. APOBEC3 confers resistance to oncolytic VSV therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A133.

Abstract Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus–antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161–73. ©2018 AACR.

Vascular protection using gene therapy can reduce the late toxicities of radiotherapy.

Intravenous infusion of oncolytic reovirus in patients leads to infection of brain tumors, infiltration by cytotoxic T cells, and up-regulation of PD-L1.

AbstractUnderstanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment-resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immunosurveillance. In the first, the role of TNFα changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)–mediated cytokine response characterized by low IL6 and elevated IFNγ, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNγ, inhibiting both NK-cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFα, which stimulated the growth of MRD tumors. Finally, therapies that blocked PD-1, TNFα, or NK cells delayed or prevented recurrence. These data show how innate immunosurveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identify therapeutic targets in patients with MRD known to be at high risk of relapse. Cancer Immunol Res; 5(11); 1029–45. ©2017 AACR.