Use of Liquid Patient Ascites Fluids as a Preclinical Model for Oncolytic Virus Activity.

The translational success of oncolytic virotherapies would benefit from the widespread use of clinically relevant ex vivo models. Malignant ascites, an accumulation of fluid in the peritoneum due to disseminated cancer, recapitulates many features of the tumor microenvironment, making it a valuable model for studying oncolytic virus activity. Here, we describe a method for the separation and storage of cellular and acellular components of malignant ascites, followed by flow cytometric characterization of the cellular fraction. We then outline a simple experiment using whole ascites to assess the activity of a bispecific T cell engager (BiTE)-expressing oncolytic adenovirus.

Author Correction: A dynamic COVID-19 immune signature includes associations with poor prognosis.

A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-01186-5.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples.

BACKGROUND: Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid "on-target off-tumour" toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. METHOD: A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor ß (FRß) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. RESULTS: T cells activated by the CD206- and FRß-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding - the first of its kind - demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. CONCLUSIONS: This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

Solid Tumor Immunotherapy with T Cell Engager-Armed Oncolytic Viruses.

Oncolytic viruses (OVs) are novel anticancer agents that combine direct cancer cell killing with the stimulation of antitumor immunity. In addition, OVs can be engineered to deliver biological therapeutics directly to tumors, offering unique opportunities to design multimodal anticancer strategies. Here, a case for arming OVs with bispecific T cell engagers (BiTEs) is put forward. BiTEs redirect the cytotoxicity of polyclonal T cells to target cells of choice, and have demonstrated efficacy against a number of hematological cancers. However, the success of BiTEs in the treatment of solid tumors appears more limited, at least in part due to: (i) poor delivery kinetics and penetration into tumors, and (ii) on-target off-tumor activity, leading to dose-limiting toxicities. Linking the production of BiTEs to OV replication provides an exciting means to restrict production to the tumor site, widen their therapeutic window, and synergize with direct oncolysis. This review summarizes progress thus far in the preclinical development of BiTE-armed OVs, and explores the possibility of cotargeting cancer cells and nontransformed stromal cells.

An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.

: Effective immunotherapy of stromal-rich tumors requires simultaneous targeting of cancer cells and immunosuppressive elements of the microenvironment. Here, we modified the oncolytic group B adenovirus enadenotucirev to express a stroma-targeted bispecific T-cell engager (BiTE). This BiTE bound fibroblast activation protein on cancer-associated fibroblasts (CAF) and CD3ε on T cells, leading to potent T-cell activation and fibroblast death. Treatment of fresh clinical biopsies, including malignant ascites and solid prostate cancer tissue, with FAP-BiTE-encoding virus induced activation of tumor-infiltrating PD1+ T cells to kill CAFs. In ascites, this led to depletion of CAF-associated immunosuppressive factors, upregulation of proinflammatory cytokines, and increased gene expression of markers of antigen presentation, T-cell function, and trafficking. M2-like ascites macrophages exhibited a proinflammatory repolarization, indicating spectrum-wide alteration of the tumor microenvironment. With this approach, we have actively killed both cancer cells and tumor fibroblasts, reversing CAF-mediated immunosuppression and yielding a potent single-agent therapeutic that is ready for clinical assessment. SIGNIFICANCE: An engineered oncolytic adenovirus that encodes a bispecific antibody combines direct virolysis with endogenous T-cell activation to attack stromal fibroblasts, providing a multimodal treatment strategy within a single therapeutic agent.

Oncolytic adenovirus expressing bispecific antibody targets T-cell cytotoxicity in cancer biopsies.

Oncolytic viruses exploit the cancer cell phenotype to complete their lytic life cycle, releasing progeny virus to infect nearby cells and repeat the process. We modified the oncolytic group B adenovirus EnAdenotucirev (EnAd) to express a bispecific single-chain antibody, secreted from infected tumour cells into the microenvironment. This bispecific T-cell engager (BiTE) binds to EpCAM on target cells and cross-links them to CD3 on T cells, leading to clustering and activation of both CD4 and CD8 T cells. BiTE transcription can be controlled by the virus major late promoter, limiting expression to cancer cells that are permissive for virus replication. This approach can potentiate the cytotoxicity of EnAd, and we demonstrate using primary pleural effusions and peritoneal malignant ascites that infection of cancer cells with the BiTE-expressing EnAd leads to activation of endogenous T cells to kill endogenous tumour cells despite the immunosuppressive environment. In this way, we have armed EnAd to combine both direct oncolysis and T cell-mediated killing, yielding a potent therapeutic that should be readily transferred into the clinic.