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As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer. Keywords: bladder cancer, coxsackievirus A21, intercellular adhesion molecule-1

The distribution of tachykinin NK(1) and NK(3) receptors in the prefrontal (Brodmann area 9) and visual cortex (Brodmann area 17) of formalin-fixed postmortem human brain tissue was studied by immunohistochemistry. NK(1)-like immunoreactivity (NK(1)-LI) was observed as a thin band at the cortical surface and dots of NK(1)-LI localised on small non-pyramidal cells and in the neuropil (layers I-III). NK(3)-LI was found in beaded fibres and cells with astrocyte-like morphology in the superficial cortical layers and white matter. Dots of NK(3)-LI were prominent in the neuropil and on pyramidal (layers III/V) and non-pyramidal (layers V/VI) cells. The NK(3)-LI was more abundant and widespread than the NK(1)-LI. This is the first report of the distribution of the NK(1) receptor in the prefrontal and visual cortex of the human brain by immunohistochemistry.

Introduction Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. Methods Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. Results Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. Conclusions Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

Aim: Coxsackievirus A21 (CAVATAKTM) is a bio-selected oncolytic immunotherapy virus. Following intratumoral (i.t) injection, CAVATAK preferential infects ICAM-1 expressing tumor cells, resulting in tumor cell lysis and a systemic immune-mediated anti-tumor response. A Phase II trial of i.t delivered CAVATAK in advanced melanoma patients has highlighted antitumor activity in both injected and distant non-injected lesions. Blockade of programmed death receptor-1 (PD-1) in patients with metastatic melanoma has resulted in substantial tumor responses via a mechanism involving reversal of tumor induced T cell suppression. We hypothesized that combination of CAVATAK and PD-1 blockade may enhance antitumor responses, potentially leading to improved clinical activity. Methods: Preclinical studies in C57BL mice were conducted to assess the antitumor activity of CAVATAK and anti-mouse PD-1 (mPD-1) mAb (2A3-IgG2a) in a B16-ICAM-1 melanoma immune competent mouse model. B16-ICAM-1 cells are murine melanoma B16 cells stably transfected to express human ICAM-1 allowing CAVATAK binding and cell infection. CAVATAK (∼108TCID50) was administered i.t, while anti mPD-1 mAb (12.5 mg/kg) was delivered via the intraperitoneal route. Following treatment of the primary cutaneous B16-ICAM-1 tumor with 8 cycles of CAVATAK injections and 4 cycles of anti-PD-1mAb, mice were then challenged with an additional subcutaneous administration of B16 cells. Results: Significant single agent antitumor activities against the primary B16-ICAM-1 tumor were observed in mice treated with either CAVATAK or anti-PD-1 mAb relative to saline controls. Furthermore, combination of CAVATAK and anti-PD-1 mAb mediated significantly greater antitumor activity and offered greater survival benefit when compared to use of either agent alone. Of particular interest was the finding that a combination of CAVATAK and anti-PD-1 mAb was able to noticeably delay the onset of palpable tumor development following B16 cell challenge when compared to all other single agent treatment regimes. Conclusions: The significant anti-tumor activity mediated by the combination of CAVATAK and anti-PD-1 mAb blockade observed in the presented murine melanoma model supports clinical evaluation of such an immunotherapeutic combination treatment regime in patients with advanced melanoma. Disclosure: D. Shafren: Author is CSO of Viralytics and holds stock; M. Quah and Y. Wong: Receives research support from Viralytics; R.H. Andtbacka: has received travel support from Viralytics; G. Au: Receives research support from Viralytics and holds stock