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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.

e14517 Background: The full potential of CAR-T cells in clinic is limited by the immunosuppressive tumour microenvironment (TME), which induces expression of exhaustion markers and limits the activity of tumour infiltrating lymphocytes (TILs) including CAR-T cells. We developed a next generation inducible Armored CAR platform (aCAR) that releases an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation, limiting payload release exclusively to the TME, thereby reducing the risk of systemic exposure. This differentiated strategy facilitates activation of CAR-T cells and TILs within the TME and has the potential for lower toxicity. Methods: A ROR1-targeting second-generation CAR containing a 41BB-CD3ζ intracellular domain was cloned into a lentivirus transfer vector alongside a prototypic anti-PD1 antibody. The CAR was under the control of a constitutively active promoter, and the PD1 targeting payload was controlled by an inducible promoter that was activated upon engagement of CAR with ROR1 on tumour cells. CAR-T cells were subject to in vitro co-culture assays with target ROR1+ tumour cells and their cytotoxic responses evaluated by flow cytometry. Levels and binding functionality of released payload were analyzed by ELISA and flow cytometry. In vivo xenograft models were performed in NSG mice with tumor growth assessed by bioluminescent imaging (BLI) and caliper measurement of the tumour volume. Results: Non-Armored CAR cells displayed potent and specific cytotoxic responses directed towards ROR1+ TNBC and NSCLC cells lines at different effector to target (E:T) ratios. The results were equivalent or superior to CARs generated with comparator anti-ROR1 antibodies, which may be due to the ROR1 CAR targeting a membrane proximal epitope within the ROR1 frizzled domain. The aCAR cells released measurable levels of anti-PD1 payload within 5 hours of binding to ROR1 on tumors and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Established PDL1+ TNBC xenograft models using the aCAR cells and comparing with non-Armored CAR cells displayed a qualitative abrogation in tumor growth by BLI, which was confirmed and shown to be significant by caliper measurement of the tumor volume. Continuing the experiment out to 3 months showed a significant survival advantage for the animals receiving aCAR. All other cohorts were terminated by day 70, however 20% of the aCAR cohort survived at day 95. Conclusions: Our next generation inducible aCAR 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 and limiting payload exposure to the site of action. This technology provided a significant survival advantage in challenging in vivo xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach.

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.

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., Highlights • Thrombin is not a major direct contributor to C5a generation during venous thrombosis in mice. • Plasmin, a protease generated in response to thrombin generation and fibrin deposition, efficiently cleaves C5 to C5a. • In an arterial thrombosis model, administration of a plasminogen activator augments C5a plasma levels. • Plasmin participates in immunothrombosis, liberating chemotactic C5a and inducing assembly of the procoagulant C5b-9. Venous and arterial thrombosis are major causes of death and morbidity. Leukocytes are early and active participants in thrombus formation, recruited partly by complement factor C5a. We examined how C5a is generated in the setting of thrombosis. In venous thrombosis in mice, we show that thrombin, a key clot-promoting enzyme, is not a major contributor to C5a generation. Rather, plasmin, a fibrinolytic enzyme formed in response to thrombin generation and clot formation, efficiently generates C5a. The findings were validated in an arterial thrombosis model in mice. These insights may be valuable in developing therapeutic strategies to limit thrombus formation.

Background Immunotherapy with chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers (BiTEs) has shown impressive efficacy against CD19+ malignancies, but translation to other tumour targets is not possible. In this regard, receptor tyrosine kinase like orphan receptor 1 (ROR1) is an attractive therapeutic target that is present on a range of haematological and solid malignancies, as well as cancer stem cells, but importantly with limited expression on normal adult tissues. Our aim, through iterative optimisation, was to generate novel humanised ROR1 single chain variable fragments (scFv) that enable efficient targeting of ROR1 on tumour cells without toxicity. Methods 13 novel anti-ROR1 antibodies were isolated after a rat immunisation programme, of which ten bound as scFvs. These fragments were engineered into a second generation CAR structure and using lentiviral vectors generated ROR1CAR T cells, imparting ROR1-specific but MHC-independent cytotoxicity. Concurrently, ROR1 scFvs were linked with a CD3 scFv to generate BiTEs—ie, small molecules able to facilitate formation of a cytotoxic synapse between T cells and ROR1+ tumour targets. Findings Co-culture of our ten novel CAR T-cell constructs with ROR1+ target cells demonstrated scFv dependent cytotoxicity. Two lead candidates were selected for humanisation to minimise immunogenicity, and of 50 variants constructed one in particular was able to target haematological and solid tumour cell lines (p vs 18, p=0··0339). Interpretation Extensive evaluation and optimisation has generated ROR1 CAR and BiTE constructs that can mediate potent effector function. Although both constructs harness the immune system, their unique characteristics provide a multifaceted platform to target a broad range of malignancies, many with considerable unmet therapeutic need such as pancreatic cancer. Ongoing work aims to determine which tumour types are best targeted by BiTEs or CAR T cells and to translate these agents to the clinic. Funding Wellcome Trust, National Institute for Health Research, NHS Blood and Transplant.

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.

Bispecific T-cell Engagers (BiTEs) are a promising immunotherapeutic approach, usually consisting of two single chain variable fragments (scFv) linked in tandem. One arm binds to tumour-associated antigens, whilst the other binds CD3 on T-cells, facilitating the formation of cytotoxic synapses leading to killing of tumour targets in a MHC and co-stimulatory molecule independent manner. Blinatumomab, the most advanced BiTE in clinic targets CD19 and has shown impressive results in patients with relapsed/refractory B cell acute lymphoid leukaemia but results in elimination of normal and malignant B lymphocytes. Receptor Tyrosine Kinase Like Orphan receptor is a surface antigen expressed on a range of haematological and solid malignancies, including Chronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), and cancer stem cell-like cells (CSC). It is however expressed at low or undetectable levels in normal tissues making it an attractive target for immunotherapy approaches. We generated a panel of novel fully humanised ROR1 antibodies and selected an optimal scFv based on ROR1 binding to use in our BiTE format. Our ROR1 BiTE (ROR1xCD3), at low concentrations, was efficient inducing T-cell mediated cytotoxicity and consequent inflammatory cytokine secretion against a panel of haematopoietic cell lines with constitutive ROR1 expression in vitro. Importantly cytotoxicity was comparable to that seen with a control CD19 BITE. Moreover, ROR1xCD3 led to significant and selective cytotoxicity of primary CLL samples. We transitioned our in vitro studies to animal models and showed that our ROR1xCD3 is able to retard growth of established SKW cells engrafted in immunocompromised mice, as detected by bioluminescence imaging. Additionally ROR1xCD3 improved survival, with the ROR1xCD3 treated cohort showing improved median survival of 50 days compared to control mice of 18 days despite only receiving a single infusion of T-cells. In summary, we have developed a novel ROR1 BiTE able to target an array of haematopoietic malignancies. Our ROR1 BiTE induces T-cell cytotoxicity against ROR1 positive cell lines in vitro and in vivo as well as primary CLL cells and has the potential to be a beneficial therapeutic for CLL. Disclosures Gohil: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies.

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia of the elderly, remains largely incurable despite chemotherapy and small molecule inhibitors. Treatment targeting CD19 and CD20 has achieved important clinical benefits; however, these antigens are also present on normal B-cells rendering the patients immunodeficient and at a higher risk of infections. In view of this, our immunotherapy strategy is to produce therapeutic antibodies targeting the receptor-tyrosine-kinase-like orphan receptor 1 (ROR1), a protein present on the surface of malignant B-cells and cancer stem cell-like cells (CSC), but absent on most normal tissue. In order to produce new ROR1 monoclonal antibodies (MAbs), we generated a rat hybridoma library and screened >150 anti-ROR1+clones. We then cloned 16 of our own novel antibodies as well as previously published clones: 4a5 and R12 to human IgG1, kappa constant regions. A total of 13 chimeric antibodies recognised human ROR1 on different cell types, out of which, clone SA1 showed superior complement-dependent cytotoxicity (CDC) than other ROR1 MAbs, including clones 4a5 and R12. To determine the binding domain of our ROR1 MAbs, we generated stable cell lines expressing either full length-ROR1 or various forms of truncated ROR1 extracellular domains. Flow cytometry evaluation using these cells, and Epitope Mapping ELISA using a ROR1 peptide library, showed that our antibodies preferentially bind the Ig-like and Frizzled domains. Additionally, we fused clone SA1 to a mouse IgG2a, kappa constant regions in order to perform competition assays amongst our ROR1 MAbs and previously reported clones. We found that there was no epitope overlap amongst any of the tested clones. Moreover, by substituting single amino acids in a relevant region within the Ig-like domain, which we previously determined to be critical for SA1 binding, we found that this epitope is unique to SA1 and, notably is not shared by other clones previously reported in the literature; including clone D10, the prototype of Cirmtuzumab. Importantly, further characterisation of clone SA1 revealed that it can get internalised by both SKW 6.4 cells and CLL cells. As a whole, we have generated and identified a novel anti-human ROR1 monoclonal antibody able to trigger specific CDC of ROR1+ cells. More than that, clone SA1 has a unique epitope and it is able to get internalised by both cell lines and CLL cells that endogenously express ROR1. Additional characterisation and functional analyses are ongoing in order to also develop this antibody as a drug-antibody conjugate (ADC). Importantly, effective targeting of ROR1 expressed on malignant cells and CSC cells but not healthy tissue would provide a safer and more effective treatment of CLL and, in turn, other ROR1+ malignancies. Disclosures Paredes-Moscosso: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Gohil:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

Introduction: Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) is a surface antigen expressed on a range of haematological and solid malignancies including Chronic Lymphocytic Leukaemia (CLL). Although expressed during embryogenesis, its virtual absence on normal adult tissues makes it an attractive target for immunotherapy, especially with Chimeric Antigen Receptor modified T-cells (CAR T-cells). We have generated novel fully humanised ROR1CAR constructs for the treatment of CLL and other ROR1 positive malignancies. Results: Following a rat immunisation programme 38 oligloclonal hybridoma clones were single cell sorted and subjected to 5'RACE. Of 13 novel anti-ROR1 antibodies isolated, 10 retained specific binding when cloned into a heavy-linker-light single chain variable fragment (scFv) format. These scFvs in combination with a second generation CAR architecture comprising CD3zeta and 4-1BB demonstrated specific toxicity against ROR1 positive cell lines after T-cell transduction using lentiviral vectors. We found cytotoxicity with ROR1CAR T-cells was dependent on target cell ROR1 density. In order to ensure our screening assays allowed us to select which of the 10 binders was most suitable for targeting primary CLL, we assessed the antigen density of ROR1 and CD19 on CLL cells. Median expression of ROR1 was 2304 molecules/cell (Range 800-4828), compared to CD19, which had a much higher density of 12,583 (Range 5894-23,652). In view of this, subsequent functional assessment was focused on SKW and Jeko1 cell lines with constitutive ROR1 expression at levels similar to CLL cells, as opposed to those transduced to express supra-physiological levels. Our initial optimisation focused on modifying the CAR extracellular spacer region. We demonstrated a reciprocal relationship between cytotoxicity and the distance between T-cells and target cells. This was assessed by using clones that bound either the membrane-distal immunoglobulin domain or a more membrane-proximal frizzled domain of ROR1. The use of an optimum spacer enhanced cytotoxicity of all scFv constructs but yielded two lead candidates: Clones A & F. These showed consistently superior cytotoxicity against target cell lines compared to the other isolated clones. In addition epitope mapping revealed binding sites unique from the previously described rabbit R12 and murine 4A5 anti-ROR1 CAR T-cells. One of the advantages of targeting ROR1 as opposed to CD19 is sparing the normal B-cell compartment from CAR mediated eradication. However this comes with the consequent risk of B-cell mediated immune responses against rat-derived scFvs. To minimise immunogenicity we undertook a humanisation programme and grafted the complementary determining regions (CDR) of the heavy and light chains of Clone A and F into 5 acceptor human germline VH and VL sequences, generating 25 potential scFvs for each. Binding assessment showed seventeen successfully humanised binders for Clone A and three for Clone F. Of these, 5/17 and 3/3 showed activity in a CAR format against target cells. A final selection was made based on specific cytotoxicity, enhanced cytokine secretion (Interleukin-2 and Interferon gamma) and proliferation compared to the parental clones resulting in 2 unique constructs targeting different extracellular domains of ROR1. In addition, we have demonstrated cytotoxicity against a panel of ROR1 positive solid cancer cell lines to demonstrate their wider applicability. Conclusion: ROR1CAR T-cells have the potential to be an effective therapeutic not just for CLL but also Acute Lymphoblastic Leukaemia, Mantle Cell Lymphoma and solid malignancies. We have described the first humanised ROR1 CARs, which target novel epitopes and have proved effective in relevant pre-clinical assays. Although other ROR1 CARs have been described, we believe the unique properties of these constructs merits further investigation and comparison in the preclinical and clinical setting. Disclosures Gohil: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Pule:Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding; Amgen: Honoraria. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.