Assembly of alternative prothrombinase by extracellular histones initiates and disseminates intravascular coagulation.

Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)- and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

Phenotypic immune characterization of gastric and esophageal adenocarcinomas reveals profound immune suppression in esophageal tumor locations.

Background: Tumors in the distal esophagus (EAC), gastro-esophageal junction including cardia (GEJAC), and stomach (GAC) develop in close proximity and show strong similarities on a molecular and cellular level. However, recent clinical data showed that the effectiveness of chemo-immunotherapy is limited to a subset of GEAC patients and that EACs and GEJACs generally benefit less from checkpoint inhibition compared to GACs. As the composition of the tumor immune microenvironment drives response to (immuno)therapy we here performed a detailed immune analysis of a large series of GEACs to facilitate the development of a more individualized immunomodulatory strategy. Methods: Extensive immunophenotyping was performed by 14-color flow cytometry in a prospective study to detail the immune composition of untreated gastro-esophageal cancers (n=104) using fresh tumor biopsies of 35 EACs, 38 GEJACs and 31 GACs. The immune cell composition of GEACs was characterized and correlated with clinicopathologic features such as tumor location, MSI and HER2 status. The spatial immune architecture of a subset of tumors (n=30) was evaluated using multiplex immunohistochemistry (mIHC) which allowed us to determine the tumor infiltration status of CD3+, CD8+, FoxP3+, CD163+ and Ki67+ cells. Results: Immunophenotyping revealed that the tumor immune microenvironment of GEACs is heterogeneous and that immune suppressive cell populations such as monocytic myeloid-derived suppressor cells (mMDSC) are more abundant in EACs compared to GACs (p<0.001). In contrast, GACs indicated a proinflammatory microenvironment with elevated frequencies of proliferating (Ki67+) CD4 Th cells (p<0.001), Ki67+ CD8 T cells (p=0.002), and CD8 effector memory-T cells (p=0.024). Differences between EACs and GACs were confirmed by mIHC analyses showing lower densities of tumor- and stroma-infiltrating Ki67+ CD8 T cells in EAC compared to GAC (both p=0.021). Discussions: This comprehensive immune phenotype study of a large series of untreated GEACs, identified that tumors with an esophageal tumor location have more immune suppressive features compared to tumors in the gastro-esophageal junction or stomach which might explain the location-specific responses to checkpoint inhibitors in this disease. These findings provide an important rationale for stratification according to tumor location in clinical studies and the development of location-dependent immunomodulatory treatment approaches.

Longitudinal immune monitoring of patients with resectable esophageal adenocarcinoma treated with Neoadjuvant PD-L1 checkpoint inhibition.

The analysis of peripheral blood mononuclear cells (PBMCs) by flow cytometry holds promise as a platform for immune checkpoint inhibition (ICI) biomarker identification. Our aim was to characterize the systemic immune compartment in resectable esophageal adenocarcinoma patients treated with neoadjuvant ICI therapy. In total, 24 patients treated with neoadjuvant chemoradiotherapy (nCRT) and anti-PD-L1 (atezolizumab) from the PERFECT study (NCT03087864) were included and 26 patients from a previously published nCRT cohort. Blood samples were collected at baseline, on-treatment, before and after surgery. Response groups for comparison were defined as pathological complete responders (pCR) or patients with pathological residual disease (non-pCR). Based on multicolor flow cytometry of PBMCs, an immunosuppressive phenotype was observed in the non-pCR group of the PERFECT cohort, characterized by a higher percentage of regulatory T cells (Tregs), intermediate monocytes, and a lower percentage of type-2 conventional dendritic cells. A further increase in activated Tregs was observed in non-pCR patients on-treatment. These findings were not associated with a poor response in the nCRT cohort. At baseline, immunosuppressive cytokines were elevated in the non-pCR group of the PERFECT study. The suppressive subsets correlated at baseline with a Wnt/ß-Catenin gene expression signature and on-treatment with epithelial-mesenchymal transition and angiogenesis signatures from tumor biopsies. After surgery monocyte activation (CD40), low CD8+Ki67+ T cell rates, and the enrichment of CD206+ monocytes were related to early recurrence. These findings highlight systemic barriers to effective ICI and the need for optimized treatment regimens.

An ROR1 bi-specific T-cell engager provides effective targeting and cytotoxicity against a range of solid tumors.

We have developed a humanized bi-specific T-cell engager (BiTE) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), a cell surface antigen present on a range of malignancies and cancer-initiating cells. Focusing initially on pancreatic cancer, we demonstrated that our ROR1 BiTE results in T cell mediated and antigen-specific cytotoxicity against ROR1-expressing pancreatic cancer cell lines in vitro at exceedingly low concentrations (0.1 ng/mL) and low effector to target ratios. Our BiTE prevented engraftment of pancreatic tumor xenografts in murine models and reduced the size of established subcutaneous tumors by at least 3-fold. To validate its wider therapeutic potential, we next demonstrated significant cytotoxicity against ovarian cancer in an in vitro and in vivo setting and T-cell-mediated killing of a range of histologically distinct solid tumor cell lines. Overall, our ROR1 BiTE represents a promising immunotherapy approach, because of its ability to target a broad range of malignancies, many with significant unmet therapeutic needs.

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin.

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin-antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

Antibody therapies for melanoma: new and emerging opportunities to activate immunity (Review).

The interface between malignant melanoma and patient immunity has long been recognised and efforts to treat this most lethal form of skin cancer by activating immune responses with cytokine, vaccine and also antibody immunotherapies have demonstrated promise in limited subsets of patients. In the present study, we discuss different antibody immunotherapy approaches evaluated in the context of melanoma, each designed to act on distinct targets and to employ different mechanisms to restrict tumour growth and spread. Monoclonal antibodies recognising melanoma-associated antigens such as CSPG4/MCSP and targeting elements of tumour-associated vasculature (VEGF) have constituted long-standing translational approaches aimed at reducing melanoma growth and metastasis. Recent insights into mechanisms of immune regulation and tumour-immune cell interactions have helped to identify checkpoint molecules on immune (CTLA4, PD-1) and tumour (PD-L1) cells as promising therapeutic targets. Checkpoint blockade with antibodies to activate immune responses and perhaps to counteract melanoma-associated immunomodulatory mechanisms led to the first clinical breakthrough in the form of an anti-CTLA4 monoclonal antibody. Novel modalities to target key mechanisms of immune suppression and to redirect potent effector cell subsets against tumours are expected to improve clinical outcomes and to provide previously unexplored avenues for therapeutic interventions.