The last few years have seen an increased interest in immunotherapy in the treatment of malignant disease. In particular, there has been significant enthusiasm for oncolytic virotherapy, with a large amount of pre-clinical data showing promise in animal models in a wide range of tumour types. How do we move forward into the clinical setting and translate something which has such potential into meaningful clinical outcomes? Here, we review how the field of oncolytic virotherapy has developed thus far and what the future may hold.

Background The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. Methods This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. Results An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. Conclusion This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations.

Pexa-Vec is an engineered Wyeth-strain vaccinia oncolytic virus (OV), which has been tested extensively in clinical trials, demonstrating enhanced cytotoxic T cell infiltration into tumours following treatment. Favourable immune consequences to Pexa-Vec include the induction of an interferon (IFN) response, followed by inflammatory cytokine/chemokine secretion. This promotes tumour immune infiltration, innate and adaptive immune cell activation and T cell priming, culminating in targeted tumour cell killing, i.e., an immunologically ‘cold’ tumour microenvironment is transformed into a ‘hot’ tumour. However, as with all immunotherapies, not all patients respond in a uniformly favourable manner. Our study herein, shows a differential immune response by patients to intravenous Pexa-Vec therapy, whereby some patients responded to the virus in a typical and expected manner, demonstrating a significant IFN induction and subsequent peripheral immune activation. However, other patients experienced a markedly subdued immune response and appeared to exhibit an exhausted phenotype at baseline, characterised by higher baseline immune checkpoint expression and regulatory T cell (Treg) levels. This differential baseline immunological profile accurately predicted the subsequent response to Pexa-Vec and may, therefore, enable the development of predictive biomarkers for Pexa-Vec and OV therapies more widely. If confirmed in larger clinical trials, these immunological biomarkers may enable a personalised approach, whereby patients with an exhausted baseline immune profile are treated with immune checkpoint blockade, with the aim of reversing immune exhaustion, prior to or alongside OV therapy.

Open-access immunosequencing data | West, Emma | TBD | Adaptive Biotechnologies | Background: Effective treatment for patients with metastatic cancer is limited, particularly for colorectal cancer patients with metastatic liver lesions (mCRC), where accessibility to numerous tumours is essential for favorable clinical outcomes. Oncolytic viruses (OVs) selectively replicate in cancer cells; however, direct targeting of inaccessible lesions is limited when using conventional intravenous or intratumoural administration routes. Methods: We conducted a multi-centre, dose-escalation, phase I study of vaccinia virus, TG6002, via intrahepatic artery (IHA) delivery in combination with the oral pro-drug 5-fluorocytosine to fifteen mCRC patients. Results: Successful IHA delivery of replication-competent TG6002 was achieved, as demonstrated by virus within tumour biopsies. Functional transcription of the FCU1 transgene indicates viral replication within the tumour, with higher plasma 5-fluorouracil associated with patients receiving the highest dose of TG6002. IHA delivery of TG6002 correlated with a robust systemic peripheral immune response to virus with activation of peripheral blood mononuclear cells, associated with a proinflammatory cytokine response and release of calreticulin, potentially indicating immunogenic cell death. Gene Ontology analyses of differentially-expressed genes reveal a significant immune response at the transcriptional level in response to treatment. Moreover, an increase in the number and frequency of T-cell receptor clones against both cancer- and neo-antigens, with elevated functional activity, may be associated with improved anti-cancer activity. Despite these findings, no clinical efficacy was observed. Conclusions: In summary, these data demonstrate delivery of OV to tumour via IHA administration, associated with viral replication and significant peripheral immune activation. Collectively, the data supports the need for future studies using IHA administration of OVs.

Introduction/Objectives: Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is critical for accurate pathological characterisation and treatment planning, as therapies advance towards personalisation. Tumour mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint blockade but its relationship with immune infiltration in HCC remains unclear, particularly across different aetiologies and within multifocal disease. Methods: This study characterises both the inter- and intra-patient heterogeneity in HCC, integrating whole-exome sequencing with detailed histopathologic and immune cell profiling, to identify implications for diagnosis and response to treatment. Samples of 54 tumours from 26 patients undergoing surgery at the Leeds Teaching Hospital NHS Trust were examined, including 10 patients with multifocal disease. The cohort includes the major UK aetiological groups. Whole-exome sequencing of tumours and matched background liver identified somatic variants. Histopathological and immunohistochemical evaluations were performed to assess tumour features and characterise the tumour immune microenvironment. Results: Marked inter-individual heterogeneity was observed across aetiological groups with variation in tumour mutation burden, genes containing somatic mutations and immune infiltration. Multifocal tumours frequently demonstrated discordant genetic and immune profiles irrespective of clonal relatedness. NeoTMB was not associated with recurrence following surgery, whereas recurrent cases demonstrated significantly higher intratumoural FoxP3+ T-cell frequencies. Conclusions: This study demonstrates substantial genetic and immune heterogeneity in HCC, including marked discordance between multifocal tumours within the same patient. Tumour mutation burden was not related to immune cell infiltration or risk of recurrence after surgery, whereas elevated intratumoural FoxP3+ regulatory T-cell frequencies were associated with recurrence. These findings suggest that the immunological composition of the tumour microenvironment may be more relevant to clinical outcome than mutational burden alone and highlight the importance of comprehensive tumour profiling to inform personalised therapeutic strategies.