Methods for detecting pathogenic antiphospholipid antibodies and identifying inhibitors

Abstract

The present invention relates to a method for determining whether a subject is suffering from an autoimmune disease, such as antiphospholipid syndrome (APS), by detecting antiphospholipid antibodies (aPL) in a sample using a novel target, lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof. Furthermore, the present invention relates to a method for identifying an inhibitor of endothelial cell protein C receptor (EPCR) function in an autoimmune disease, preferably one that does not have side effects on the EPCR-regulating function in blood coagulation, and a method for producing a pharmaceutical composition, the method comprising the steps of identifying a candidate inhibitor and appropriately formulating the candidate inhibitor into a pharmaceutical composition. Furthermore, the present invention relates to the identified inhibitor or pharmaceutical composition for use in the prevention and / or treatment of an autoimmune disease, such as antiphospholipid syndrome, in a subject. Furthermore, the present invention relates to a method for preventing and / or treating an autoimmune disease, such as antiphospholipid syndrome, in a subject.

Description

[Technical Field]

[0001] The present invention relates to a method for determining whether a subject is suffering from an autoimmune disease, such as antiphospholipid syndrome (APS), by detecting antiphospholipid antibodies (aPL) in a sample using a novel target, lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof. Furthermore, the present invention relates to a method for identifying inhibitors of endothelial cell protein C receptor (EPCR) function in autoimmune diseases, preferably without side effects on EPCR-regulating function in blood coagulation, and a method for producing a pharmaceutical composition, comprising the steps of identifying a candidate inhibitor and appropriately formulating the candidate inhibitor into a pharmaceutical composition. Furthermore, the present invention relates to the identified inhibitor or pharmaceutical composition for use in the prevention and / or treatment of an autoimmune disease, such as antiphospholipid syndrome, in a subject. Furthermore, the present invention relates to a method for treating and / or preventing an autoimmune disease, such as antiphospholipid syndrome, in a subject. [Background technology]

[0002] Antiphospholipid syndrome (APS) is an acquired autoimmune disease in which inadequate control of the immune system leads to an increased tendency for blood to clot. The resulting blood clot (thrombosis) can subsequently lead to reduced blood flow to affected tissues (ischemia), potentially resulting in complications such as stroke, heart attack, or miscarriage. Although lipid-reactive antibodies also appear transiently in infectious diseases, the clonal evolution of persistent antiphospholipid antibodies (aPL) in autoimmune diseases causes severe thromboembolic events, recurrent pregnancy loss, and fetal death in APS (1).

[0003] Although reactivity with cardiolipin has been used to identify aPL, aPL recognizes a variety of anionic phospholipids and blood proteins, including β2-glycoprotein I (β2GPI). This complex reactivity has hindered the definition of the precise mechanisms underlying the spectrum of APS-related pathologies (1, 2) and the development of autoimmune diseases (3, 4). Although clonal expansion of monoclonal aPL leads to protein cross-reactivity (5), lipid recognition is sufficient to cause pregnancy complications (6) and thrombosis in mice (7), both of which involve crosstalk between innate immune defense complement and coagulation pathways (6, 8).

[0004] By binding to EPCR expressed by myeloid cells, aPL targets a key toggle switch controlling coagulation and innate immune signaling. PAR2 activation by the TF-FVIIa-FXa-EPCR complex supports TLR4-mediated induction of interferon-regulated genes (16) but attenuates TF-dependent PAR2 signaling by competing with the anticoagulant activating protein C-FV-protein S complex for EPCR ligand occupancy (37). Deregulated interferon signaling drives autoimmunity, and although direct targeting of EPCR aPL induces an interferon signaling response in myeloid cells, mice in which the EPCR signaling pathway has been abolished are protected from the development of autoimmune aPL.

[0005] Genetic or pharmacological inhibition of the antigen target EPCR-LBPA attenuates aPL-induced pathology in mice. Engagement of aPL with EPCR expressed on innate immune cells induces an interferon-regulated antibacterial response, driving interferon-dependent B cell proliferation and the development of autoimmunity. Specifically, aPL recognizes a single lipid-protein receptor complex required for the pathogenesis and complications of this autoimmune disease.

[0006] Patent Document 1 relates to a method for detecting autoantibodies by detecting and quantifying in vitro autoantibodies against endothelial cell protein C / activated protein C receptor (EPCR) in a sample.

[0007] Sorice et al. (Non-Patent Document 1) have disclosed that anti-LBPA antibodies and IgG from APS patients affect the distribution of intracellular β2GPI in endothelial cell cultures and in the coagulation system. Furthermore, Sorice et al. suggest that LBPA is a target of aPl and is involved in the immunopathogenesis of APS.

[0008] Alessandri et al. (Non-Patent Document 2) describe LBPA antibodies as a biomarker for patients with antiphospholipid syndrome.

[0009] Olivieri et al. (Non-Patent Document 3) reviewed the clinical value of anti-LBPA and revealed that anti-LBPA antibodies cannot be used to diagnose APS.

[0010] The above-mentioned prior art discloses that endothelial cell protein C receptor (EPCR) and lysobisphosphatidic acid (LBPA), or antibodies directed against endothelial cell protein C receptor (EPCR) or lysobisphosphatidic acid (LBPA), can be used as biomarkers for diagnosing APS in patients. However, it is disclosed that antibodies against lysobisphosphatidic acid (LBPA) have no advantages as biomarkers compared to the analysis of other antibodies, such as antibodies against cardiolipin. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent Application Publication No. 2007-0141625 [Non-patent literature]

[0012] [Non-Patent Document 1] Evidence for anticoagulant activity and beta2-GPI accumulation in late endosomes of endothelial cells induced by anti-LBPA antibodies. Thromb Haemost. 2002 Apr;87(4):735-41. PMID: 12008959 [Non-patent document 2] Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. Clin Exp Immunol. 2005 Apr;140(l):173-80. doi: 10.1111 / j.1365-2249.2005.02727.x. PMID: 15762889 [Non-patent document 3] Clinical value of antibodies to lysobisphosphatidic acid in patients with primary antiphospholipid syndrome. Reumatismo. 2010 Apr-Jun;62(2):107-12. Italian. doi: 10.4081 / reumatismo.2010.107. PMID: 20657887 Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, it is an object of the present invention to provide a reliable and robust method for detecting autoimmune diseases, such as antiphospholipid syndrome (APS), particularly primary or secondary APS, based on the binding of antiphospholipid antibodies (aPL).

[0014] It is a further object of the present invention to provide methods for identifying candidate inhibitors that block aPL pathogenic signaling.

[0015] Another object is to provide a method for preparing a pharmaceutical composition, which includes, inter alia, such an inhibitor.

[0016] Yet another object of the present invention is to provide a method for treating and / or preventing an autoimmune disease, such as antiphospholipid syndrome (APS), particularly primary or secondary APS, in a subject by administering to the subject a pharmaceutical composition containing the inhibitor.

[0017] Other aspects and objects will become apparent to those skilled in the art upon review of the following description of the invention. [Means for solving the problem]

[0018] Surprisingly, in the context of the present invention, the inventors identified endosomal lysobisphosphatidic acid (LBPA) and its presentation by CD1d-like endothelial cell protein C receptor (EPCR) as the previously unknown disease-causing cell surface antigen recognized by aPL. Intersecting with the innate immune and coagulation signaling functions of EPCR, aPL engages EPCR for endosomal trafficking and initiation of prothrombotic and proinflammatory signaling.

[0019] The present inventors have further succeeded in demonstrating that the interaction between endothelial cell protein C receptor (EPCR) and LBPA is important in the process of antiphospholipid syndrome. This surprising discovery makes it possible to use the EPCR-LBPA complex as a novel target for diagnostic screening procedures and for the generation of drugs that can be used to treat and prevent antiphospholipid syndrome.

[0020] In a first aspect, the present invention solves the above problem by providing a method for determining whether a subject is suffering from an autoimmune disease, the method comprising detecting binding of antiphospholipid antibodies (aPL) in a biological sample obtained from the subject to lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof, wherein the binding of aPL to lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment thereof detects the autoimmune disease in the subject.

[0021] In a second aspect, the present invention relates to a method for identifying inhibitors of endothelial cell protein C receptor (EPCR) function / activity in autoimmune diseases, which preferably do not interfere with its function in coagulation, comprising providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof, contacting a candidate inhibitor with the sample, testing the binding of LBPA to the EPCR protein or the LBPA-binding fragment thereof in the presence or absence of the candidate inhibitor, and identifying the candidate inhibitor based on the LBPA binding as tested.

[0022] In a third aspect, the present invention relates to a method for identifying inhibitors of endothelial cell protein C receptor (EPCR) function in autoimmune diseases, which preferably do not interfere with the regulatory function of EPCR in coagulation, comprising providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding portion thereof, allowing LBPA to bind to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA complex, contacting a candidate inhibitor with said sample, testing the binding or cellular effect / function of antiphospholipid antibodies (aPL) in the presence or absence of said candidate inhibitor, and identifying said candidate inhibitor based on interference with said aPL binding or cellular function as tested.

[0023] In a fourth aspect, the present invention relates to a method for producing a pharmaceutical composition, the method comprising the steps of identifying a candidate inhibitor or inhibitor as described herein and suitably formulating said candidate inhibitor or inhibitor into a pharmaceutical composition.

[0024] In a fifth aspect, the present invention relates to an inhibitor as identified, or a pharmaceutical composition as described herein, for use in the prevention and / or treatment of an autoimmune disease in a subject.

[0025] In a sixth aspect, the present invention relates to a method for treating and / or preventing an autoimmune disease, such as antiphospholipid syndrome, in particular primary or secondary APS, in a subject, comprising administering to said subject in need of such treatment and / or prevention an effective amount of an inhibitor as identified and described herein, or a pharmaceutical composition as described herein. [Brief explanation of the drawings]

[0026] [Figure 1-1]EPCR is a receptor for aPL. (A) EPCR-dependent induction of IFN-regulated genes in monocytes by LPS and IgG from a patient infected with Treponema pallidum. (B) Induction of IFN-regulated genes by aPL. (C) Induction of TF and Tnfα mRNA and early ROS production by 3-hour stimulation of CD115+ splenocytes from the indicated mice with aPL HL5B or HL7G; mean ± SD, n = 6; *p < 0.0001; one-way ANOVA, Dunnett's multiple comparison test. (D) Live-cell imaging of HL5B internalization in monocytes from the indicated mouse strains. Bar = 5 μm. (E) Live-cell imaging of colocalization of aPL HL5B Fab'2 or IgG with EPCR using non-inhibitory αEPCR 1489 in human MM1 cells. (F) Internalization of EPCR, FVIIa, and TF in MM1 cells stimulated for 15 min similarly required protease and integrin trafficking. To quantitate internalization, surface staining was quenched with 0.4% trypan blue; mean ± SD, n = 6, *p < 0.0001; one-way ANOVA, Dunnett's multiple comparison test compared to IgG control. [Figure 1-2] Same as above [Figure 2] Figure 1 shows that EPCR is required for aPL signaling. (A) Summary of the functional properties of αEPCR relative to human and mouse EPCR. (B, C) TNF and TF induction in primary monocytes (B) and MM1 cells (C) stimulated with HL5B or HL7G for 3 hours and pretreated with anti-human EPCR antibody for 15 minutes; mean ± SD, n = 6. (D) CD115+ splenocytes of the indicated mouse strains and (E) trophoblast induction of TNFα after stimulation with IgG (100 μg / ml) isolated from APS patients for 1 hour or 3 hours, which showed cardiolipin reactivity (αCL) only, αβ2GP reactivity only, or dual reactivity. Human trophoblasts were pretreated with either non-inhibitory αEPCR 1489 or inhibitory αEPCR 1496. [Figure 3-1]Figure 1 shows that EPCR displays late endosomal lysobisphosphatidic acid (LBPA) on the cell surface. (A) Effect of aPL HL5B, aPL HL7G, and αEPCR antibodies on EPCR-dependent aPC production in mouse microvascular endothelial cells. (B) Effect of anti-mouse EPCR antibodies 1682 and 1650 on TNFα mRNA induction by aPL HL5B and HL7G; mean ± SD, n = 6, *p < 0.0001; one-way ANOVA, Dunnett's multiple comparisons. (C) Effect of αEPCR on aPL HL5B and HL7G internalization into CD115+ splenocytes. Bar = 5 μm. (D) Flow cytometric detection of FXa and EPCR in CD115+ splenic monocytes isolated from the indicated mouse strains. (E) Effect of 10 μM LBPA pretreatment for 10 min on surface binding of αEPCR 1682 and αLBPA 6C4 to the indicated monocytes; mean ± SD, n = 6, *p < 0.003; multiple t-test. (F) Competition of αEPCR 1650 and 1682 with FITC-labeled anti-LBPA antibody 6C4 binding to mouse CD115+ splenocytes. (G) Competition of αLBPA 6C4 with αEPCR 1682 binding to mouse monocytes. (H) Effect of LBPA, cardiolipin (CL), and phosphatidylserine (PS) (10 μM) on aPL HL5B signaling in EPCR C / S monocytes. Shown after 3 h of TNF induction; mean ± SD, n = 6. (I) LBPA loading of purified mouse or human sEPCR, as evidenced by faster migration on native gels. (J) Binding of aPL HL5B to purified human sEPCR or sEPCR-LBPA was analyzed by surface plasmon resonance. Because there was no apparent cooperative binding, affinity calculations were based on a monovalent binding model. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4-1]Figure 1 shows the effect of LBPA loading on aPL interaction. (A) Competition between LBPA-loaded or unmodified sEPCR and FITC-labeled HL5B Fab'2 fragment or control for binding to mouse monocytes was measured by flow cytometry. (B) LBPA-loaded EPCR is a more potent inhibitor of aPL HL5B signaling than unmodified EPCR. (C) LBPA loading of human sEPCR does not alter its competition for aPC production in mouse endothelial cells. (D) HL5B binding to control CHO cells and CHO cells expressing mouse EPCR (mEPCR). Cells were untreated or preincubated with 10 μM LBPA for 30 min; mean ± SD, n = 6. (E) Binding of anti-β2GPI aPL rJGG9 or control IgG to CHO cells transfected with mouse EPCR was measured by a fluorescence microplate reader; mean ± SD, n = 3. (F) Binding of aPL HL5B, aPL HL7G, or control IgG to CHO cells transfected with mouse (mEPCR) or human (hEPCR). Cells were loaded with 10 μM LBPA for 30 min before staining. (G) Binding of aPL HL5B (left panel) or HL7G (right panel) to LBPA-loaded mouse EPCR after 15 min of preincubation with various concentrations of purified sEPCR, either unmodified or LBPA-loaded; mean ± SD, n = 6. (H) Dose-response curves of PS exposure induced by HL5B and HL7G, measured by Annexin 5 surface staining. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 5-1]These figures show that aPL promotes EPCR-LBPA activation of cell surface acid sphingomyelinase and thrombosis. (A) aPL-mediated TF activation, PS exposure as measured by Annexin 5 staining, ROS production, and TNFα induction, and (B) aPL internalization in MM1 cells were blocked by the sphingomyelinase inhibitor desipramine. Bar = 5 μm. (C) aPL-induced ASM activity in MM1 cells is blocked by FXa, thrombin, and an inhibitor of PAR1 cleavage. (D) Live-cell imaging of surface ASM exposure in MM1 cells after 30 minutes of stimulation with Fab'2 aPL HL5B. Bar = 5 μm. (E) ASM activity in unstimulated cell lysates is blocked by αEPCR 1682 after the addition of sEPCR-LBPA (2.5 μM). For all ASM activity assays: mean ± SD, n = 3, *p < 0.0003; one-way ANOVA, Dunnett's multiple comparison test. (F) HL5B-induced thrombosis was analyzed in the flow-restricted inferior vena cava of WT mice treated with the indicated αEPCR antibodies. (G, H) Thrombosis induction by the dual-reactive aPL HL7G in the presence of the indicated αEPCR antibodies in the indicated mouse strains or WT mice. (F-H) Quantification of thrombus dimensions 3 hours after aPL induction; median, interquartile range, and range; n = 6-11; *p < 0.004; compared to αEPCR 1650 by one-way ANOVA with Dunnett's multiple comparison test. (I, J) Thrombosis induction by aPL HL5B (I) or IgG isolated from age-matched 16-week-old lupus-prone MRL / lpr and MRL control mice (J) in the indicated mouse strains. Quantification of thrombus dimensions 3 hours after aPL induction; median, interquartile range, and range; (I) n = 6-10; *p = 0.001; unpaired t-test. (J) n = 5; *p = 0.0025; two-way ANOVA, Sidak's multiple comparison test. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 6]These figures show that aPL promotes EPCR-LBPA activation of cell surface acid sphingomyelinase. (A) Induction of ASM activation by WT CD115+ splenic monocytes after 15 min of stimulation of aPL HL5B with the indicated inhibitors. (B) Loading of EPCR C / S cells with LBPA (10 μM) enabled ASM activation in HL5B-stimulated CD115+ monocytes. (C) aPL HL5B did not activate ASM in TfpiΔK1 cells. However, thrombin (1 U / ml) activation of ASM in WT and TfpiΔK1 cells was blocked by αEPCR 1682 but not by αEPCR 1650. [Figure 7] aPL-EPCR signaling promotes fetal lethality. (A) TNFα mRNA induction after 2 hours with HL5B is blocked in Alix-deficient trophoblasts; mean ± SD, n = 6, *p < 0.0001; t-test after Shapiro-Wilk test for normal distribution. (B, C) Proximity ligation assays (PLA) of ASM and EPCR performed on scrambled control JAR or ALIX- / - cells after 10 minutes of stimulation with HL5B (B) or thrombin (C), with or without LBPA loading. Bar = 25 μm. (D) aPL internalization in ALIX-deficient JAR cells and EPCR signaling in C / S monocytes (E) were restored by adding 10 μM LBPA (S,R), but not other phospholipids. (F) Pregnancy loss was scored at 15.5 days postcoitum after injection of aPL HL5B on days 8 and 12. *p<0.02; one-way ANOVA, Dunnett's multiple comparison test. (G) Schematic of aPL signaling leading to thrombosis or pregnancy complications. [Figure 8]Figure 1 shows that EPCR-LPBA is required for aPL signaling in trophoblast cells. (A) WB analysis of ALIX-deficient JAR cells. (B) Reduced LBPA surface expression in ALIX-knockdown trophoblast (JAR) cells expressing EPCR. Cells were stained with FITC-labeled αEPCR or αLBPA antibodies, and antibody surface binding was detected using a microplate fluorometer. (C) Proximity ligation assay (PLA) of ASM and EPCR performed on scrambled control JAR cells after 10 min of stimulation with thrombin and HL5B, with or without the thrombin inhibitor hirudin. Bar = 25 μm. [Figure 9] Figure 1 shows that EPCR is required for aPL interferon signaling and the proliferation of lipid-reactive aPL-producing B cells. (A) Gbp2 mRNA induction in EPCR C / S or WT monocytes after 1 hour of stimulation with HL5B, HL7G, or LPS (100 ng / ml) with or without LBPA. (B) WT monocytes were stimulated for 1 hour with IgG isolated from MRL / lpr lupus-prone mice or control MRL mice in the presence of the indicated antibodies against EPCR. (C) Human monocyte-derived DCs were cocultured with B cells in the presence of the TLR7 / 8 agonist R848 and aPL HL5B plus the indicated antibodies against human EPCR. After 10 days, anticardiolipin titers were determined. (D–F) Cocultures of splenic plasmacytoid dendritic cells (pDCs) and B cells isolated from the indicated mouse strains were cocultured with the Tlr7 agonist R848 and aPL HL5B for 10 days, followed by determination of anticardiolipin titers. IFNR− / −, type I interferon receptor-deficient mice. [Figure 10-1]EPCR signaling drives aPL expansion in vivo. (A, B) Mice of the indicated genotypes were immunized with aPL HL5B or isotype-matched control IgG, and serum anticardiolipin titers were determined at the indicated time points. (C) Cells reactive with negatively charged liposomes were detected only in mice immunized with aPL HL5B, but not with isotype-matched IgG. EPCR-LBPA, but not EPCR, competed with ribosomes for binding to these CD19+CD5+CD43+CD27+ memory B1a cells. (D) Immunization with human β2GPI induced similar high-titer IgG antibody responses to human β2GPI in EPCRWT and EPCRC / S mice. (E) Antibody titers to LBPA were detected only in EPCRWT mice; antibody titers to mouse prothrombin were not detected, and were not detected in EPCRC / S mice after five immunizations with human β2GPI. (F) IgG from human β2GPI-immunized EPCRWT, but not EPCR, mice induced monocyte TF activity and pro-inflammatory signaling in monocytes. [Figure 10-2] Same as above [Figure 11-1] Figure 1 shows the therapeutic relevance of intervention in the EPCR-LBPA pathway in exemplary settings of autoimmunity and lupus erythematosus. (A) MRL-Faslpr lupus-prone mice were treated with the indicated αEPCR antibodies at 4 weeks of age (day 0), and serum anticardiolipin titers were determined at the indicated time points; n = 5, *P = 0.03; **P < 0.0001; two-way ANOVA, Sidak's multiple comparison test. (B) Antibodies to double-stranded (ds) DNA were measured 2 weeks after the final dose in αEPCR 1650- and αEPCR-LBPA 1682-treated MRL-Faslpr mice or in 6-week-old MRL / MpJ control or MRL-Faslpr mice; n = 4-5, *P < 0.0001. (C) Immune cell infiltration in αEPCR-treated MRL-Faslpr mice; n = 5, *P < 0.025. (D) Kidney pathology scores in αEPCR-treated MRL-Faslpr mice; n = 5, *P = 0.0317; Mann-Whitney U test. [Figure 11-2] Same as above [Figure 12-1] These figures show that EPCR-LBPA is required for the development of autoimmune diseases. (A) Reactivity of purified IgG (40 μg / ml) from MRL / MpJ control mice and MRL-Faslpr mice treated with αEPCR 1650 or αEPCR-LBPA 1682 with immobilized LBPA or cardiolipin; n = 6–7, *P < 0.0001, difference from control αEPCR 1650-treated mice; two-way ANOVA, Sidak's multiple comparison test. (B) Infiltration of CD45+ / F4 / 80+ immune cells into the kidneys of MRL-Faslpr mice treated with non-inhibitory αEPCR 1650 or inhibitory αEPCR-LBPA 1682; n = 5–7, * = 0.024. (C) The phenotype of F4 / 80+ cells in the kidneys of MRL-Faslpr mice was determined by cytokine staining. (D) Albuminuria in MRL / MpJ control mice and MRL-Faslpr mice treated with the indicated antibodies for 6 weeks starting at 4 weeks of age. [Figure 12-2] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0027] The following describes the components of the present invention. While these components are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to form further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those explicitly described embodiments. This description should be understood to support and encompass embodiments combining two or more explicitly described embodiments, or combining one or more explicitly described embodiments with any number of disclosed and / or preferred components. Furthermore, all permutations and combinations of all components described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0028] As described above, in a first aspect, the present invention relates to a method for determining whether a subject is suffering from an autoimmune disease, comprising detecting binding of antiphospholipid antibodies (aPL) to lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof in a biological sample obtained from the subject, wherein the binding of aPL to lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof detects the presence of an autoimmune disease in the subject.

[0029] "LBPA-binding fragment," as used herein, is intended to mean a portion or fragment of endothelial cell protein C receptor (EPCR) such that the LBPA-binding fragment is still capable of binding, preferably, lysobisphosphatidic acid (LBPA), i.e., the receptor affinity of endothelial cell protein C receptor (EPCR) is maintained in the LBPA-binding fragment. Also included are structural mimetics of such binding domains. Preferably, all or part of the LBPA-binding fragment is produced recombinantly in a suitable expression system or by chemical synthesis.

[0030] Although APS is the only manifestation of autoimmunity in many patients (primary APS), APS also occurs in the context of other autoimmune diseases, particularly systemic lupus erythematosus (SLE) (secondary APS). Thus, a preferred embodiment of the present invention is when the autoimmune disease is antiphospholipid syndrome, particularly primary or secondary APS. Additional autoimmune diseases are selected from, but are not limited to, primary Sjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

[0031] As used herein, the term "antiphospholipid antibodies (aPL)" refers to autoantibodies that generally bind to negatively charged phospholipids, including cardiolipin (CL) as an antigen. Also included are antigen-binding fragments of such antibodies (see below for further explanation).

[0032] As used herein, the terms "binding" of aPL to the LBPA bound to EPCR or its LBPA-binding fragment, or binding of LBPA to EPCR or its LBPA-binding fragment, or further intermolecular binding between molecules in the context of the present invention, are based on non-covalent interactions. These "non-covalent" interactions refer to chemical interactions between atoms that do not share electron pairs. Non-covalent interactions are classified as hydrogen bonds, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. The existence of binding between each of the binding partners is examined using a "binding assay" based on the individual interactions of the specific binding between the binding partners. Suitable binding assays are known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0033] Further preferred is an embodiment of the present invention, wherein said lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof is immobilized, preferably directly or indirectly, to a solid support material.

[0034] As used herein, the term "direct" immobilization refers to the immobilization of isolated soluble endothelial cell protein C receptor (EPCR) or an isolated soluble LBPA-binding fragment, in which lysobisphosphatidic acid (LBPA) binds to the endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment, and the endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment is directly covalently immobilized to a solid support material, for example, via a photochemical method. To covalently immobilize biomolecules on a solid support material in a parallel and directional manner, so-called photolinkers can be used, which are bound to the endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment. The photoreaction is induced by UV irradiation, and in this case, a wavelength range above 300 nm must be used to avoid photodecomposition of the biomolecule. The photolinker reacts with a substrate for a photoinduced radical reaction, and the endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment is directly immobilized to the solid support material.

[0035] The term "indirectly" immobilized refers to immobilization of cells expressing endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment, or a portion of cells presenting endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment on their cell surface, to a solid support material, in which lysobisphosphatidic acid (LBPA) is either already bound to the endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment, or is added to the cell culture supernatant so that the LBPA-bound endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment is provided via the surface of the immobilized cells or their portions, in which case the cells or their portions are immobilized on a solid support material. The term "cell" as used in the context of the present invention refers to a eukaryotic cell capable of expressing endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment. Thus, the PROCR gene encoding endothelial cell protein C receptor (EPCR) or a nucleic acid encoding an LBPA-binding fragment can either already be present in the cell, or can be transfected into the cell with the nucleic acid or a vector containing the nucleic acid. The term "eukaryote" includes yeast cells, higher plant cells, insect cells, and mammalian cells. Once a nucleic acid or vector has been introduced into the relevant cell, the cell is maintained under conditions suitable for high level expression of the nucleic acid or vector.

[0036] The term "solid support material" refers to any solid support material that is chemically inert and allows for the direct or indirect immobilization of endothelial cell protein C receptor (EPCR) or LBPA-binding fragments thereon. Large-area immobilization can be achieved by using highly porous materials. Furthermore, the support must allow the inflow and outflow of the substances used in the context of the present invention. Several suitable supports are known. The solid support material can be selected from, for example, but not limited to, glass, agarose, polymers, or metals.

[0037] In a second aspect, the present invention relates to a method for identifying inhibitors of endothelial cell protein C receptor (EPCR) function in autoimmune diseases, preferably without interfering with the regulatory function of EPCR in blood coagulation, comprising providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof, contacting a candidate inhibitor with the sample, testing the binding of LBPA to the EPCR protein or the LBPA-binding fragment thereof in the presence or absence of the candidate inhibitor, and identifying the candidate inhibitor based on the tested LBPA binding. Thus, the assay is aimed at identifying inhibitors of the binding between LBPA and EPCR protein.

[0038] In a third aspect, the present invention relates to a method for identifying inhibitors of endothelial cell protein C receptor (EPCR) function in autoimmune diseases that do not interfere with EPCR's regulatory function in blood coagulation, comprising providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof, allowing LBPA to bind to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA complex, contacting candidate inhibitors with said sample, testing antiphospholipid antibody (aPL) binding or cellular function in the presence or absence of said candidate inhibitor, and identifying said candidate inhibitor based on interference with said aPL binding or cellular effect / function as tested. Thus, the assay seeks to identify inhibitors of binding between the LBPA / EPCR protein complex and aPL, as well as "general" inhibitors that interfere with signaling pathways involving said complex and aPL.

[0039] In addition to "binding" as described above, inhibitor candidates can also be identified through "cellular function" within intact cells present in a biological sample. "Cellular function," as used in the context of the present invention, is based on changes in protein expression of interferon-inducible genes in the cells present in the biological sample due to the presence or absence of an inhibitor candidate. For example, both inhibitory and non-inhibitory binding partners can bind to the LBPA-EPCR complex, EPCR, or aPL. Binding of the inhibitory binding partner, i.e., the inhibitor candidate, blocks the aPL-induced interferon response, while binding of the non-inhibitory binding partner does not alter the aPL-induced interferon response. The interferon response then leads to proliferation of aPL-producing B cells and expression of interferon-inducible genes. Interferon-inducible genes include, but are not limited to, IRF8, GBP2, and GBP6.

[0040] Preferred are embodiments of the methods according to the invention, wherein at least one of EPCR, fragment, LBPA, said inhibitor candidate, and / or aPL is suitably labeled and / or immobilized.

[0041] The term "suitably labeled" as used herein means that at least one of the EPCR, fragment, LBPA, the inhibitor candidate, and / or aPL may have an additional marker, such as a non-protein molecule, e.g., a nucleic acid, a sugar, or a radioactive or fluorescent marker. The label is either directly or indirectly involved in generating a detectable signal.

[0042] In another preferred embodiment of the present invention, the method further comprises testing the identified candidate inhibitors for whether they are inhibitors of endothelial cell protein C receptor (EPCR) function in autoimmune disease without interfering with EPCR function as a coagulation regulator. The inventors have shown that binding of aPL to the EPCR-LBPA complex leads to internalization of the complex and pathogenic aPL signaling. The important function of EPCR as a coagulation regulator is maintained because EPCR binds to its agonist protein C even in the absence of LBPA, and in this case, the binding of protein C to EPCR is not blocked by the identified inhibitor. This testing can also include other components of the system, LBPA and / or aPL.

[0043] In the context of the present invention, the term "suitably test" is used to distinguish between a suitable test for binding and a suitable test for cellular function. A suitable test for binding means using a suitable detection system, depending on the label used, to detect the generated detectable signal and determine whether the candidate inhibitor can inhibit the binding of LBPA to EPCR or an LBPA-binding fragment, or whether the candidate inhibitor can inhibit the binding of aPL to the LBPA-EPCR complex. For example, a suitable test can use a FRET probe, in which one binding partner is labeled with a donor fluorescent dye and the other binding partner is labeled with an acceptor fluorescent dye. The emitted fluorescent signal can be used to highly specifically detect whether the identified candidate inhibitor inhibits the binding of the relevant binding partner. Many other detection systems are known in the art. A suitable test for cellular function refers to the detection of an aPL-induced interferon response or the detection of aPL expressed by B cell proliferation. Detection can be performed by quantifying mRNA or protein at either the post-transcriptional or post-translational level. Those skilled in the art are familiar with mRNA and protein analysis methods.

[0044] Further preferred is the embodiment of the method according to the present invention, wherein said inhibitor candidate is selected from small molecules, proteins, peptides, antibodies or antigen-binding fragments thereof, enzymes, and aptamers.

[0045] The term "small molecule," as used herein, describes a class of low-molecular-weight substances not exceeding about 900 daltons. Small molecules are able to enter cells in part due to their small size. Small molecules can be chemically synthesized. The term encompasses a highly heterogeneous group of substances. Small molecules have a wide variety of biological functions, including, for example, signaling molecules. Small molecules can be of natural (e.g., secondary metabolite) or artificial (e.g., antiviral) origin. Some small molecules can cross the blood-brain barrier.

[0046] The term "protein" is used to refer to a polymer composed of amino acid monomers joined by peptide bonds. This term refers to the amino acid chain, not to a specific length of the product, and can be modified, if necessary, in vivo or in vitro, for example, by glycosylation, amidation, carboxylation, or phosphorylation. Amino acid chains shorter than about 100 amino acids in length are referred to as "peptides." The terms "peptide" and "protein" are included within the definition of "polypeptide" as used herein. A "peptide bond" is a covalent bond between two amino acids, connecting the α-amino group of one amino acid with the α-carboxyl group of the other amino acid. All amino acid or polypeptide sequences are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) unless otherwise specified.

[0047] The terms "antibody" and "antibodies" refer to antigen-binding proteins that arise in the context of the immune system. The term "antibody," as used herein, includes whole antibodies, full-length antibodies, and any fragment or derivative thereof, in which the "antigen-binding portion" or "antigen-binding region" or a single chain thereof is retained, such as the binding domain of an antibody specific for lysobisphosphatidic acid (LBPA), endothelial cell protein C receptor (EPCR), an LBPA-binding fragment, an LBPA-EPCR complex, or an antiphospholipid antibody (aPL). Naturally occurring "antibodies" (immunoglobulins) are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The heavy chain constant region is composed of three domains, CHI, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions termed complementarity-determining regions (CDRs), with more conserved regions termed framework regions (FRs) interspersed between the CDRs. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chains form two regions: the Fab (Fragment, antigen-binding) region, also known as the variable (Fv) region, and the Fc (Fragment, crystallizable) region. The variable regions (Fv) of the heavy and light chains contain the binding domain that interacts with antigen. The constant (Fc) region of the antibody may mediate binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "Fc," as used herein, includes native and variant polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Fusion proteins containing Fc portions (and oligomers formed therefrom) offer the advantage that they are easily purified by affinity chromatography over protein A or protein G columns. One suitable Fc polypeptide is derived from a human IgG1 antibody.

[0048] Fragments, derivatives, or analogs of antigen-binding proteins such as antibodies can be readily prepared using techniques known in the art. The term "antigen-binding fragment," as used herein, refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length antigen-binding protein. Examples of fragments of antigen-binding proteins encompassed by the term "antigen-binding fragment" include: a Fab fragment; a monovalent fragment consisting of the VL, VH, CL, and CHI domains; an F(ab')2 fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of the VH domain; an isolated complementarity-determining region (CDR); and a single-chain variable fragment (scFv). An antigen-binding protein, or a fragment or derivative thereof, or a fusion protein thereof, may have one or more binding sites. When two or more binding sites are present, the binding sites may be identical to or different from each other. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, whereas a "bispecific antibody" or "bifunctional antibody" has two different binding sites. Bispecific antibodies are preferred molecules of the present invention and can be selected from any bispecific format known to those skilled in the art, e.g., bite antibodies or diabodies. A "derivative" of an antigen-binding protein is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation with another chemical moiety (e.g., polyethylene glycol or albumin, such as human serum albumin), phosphorylation, and / or glycosylation.

[0049] An "scFv" is a monovalent molecule that can be engineered using recombinant methods by joining the two domains of the Fv fragment, VL and VH, with a synthetic linker that can combine them into a single protein chain. Such single-chain antigen-binding peptides are also intended to be encompassed within the term "antigen-binding protein." These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0050] The terms "antigen-binding fragment" or "antigen-binding region" of an antigen-binding protein such as an antibody, or grammatical equivalents, as used herein, refer to that region or portion that confers antigen specificity. Thus, a fragment of an antigen-binding protein includes one or more fragments of the antigen-binding protein that retain the ability to specifically bind to an antigen (e.g., an HLA-peptide complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0051] The term "enzyme" as used herein refers to a protein with catalytic activity.

[0052] As used herein, the term "aptamer" refers to a short, single-stranded DNA or RNA oligonucleotide (25-70 bases) capable of binding to a specific molecule. Aptamers generally include RNA molecules, single-stranded DNA molecules, modified RNA molecules, or modified DNA molecules. The preparation of aptamers is known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding moieties.

[0053] Preferred is an embodiment of the method according to the present invention, wherein said subject is a mammal, preferably a human.

[0054] Further preferred is the embodiment of the method according to the present invention, wherein said biological sample is selected from body fluids including blood, serum and saliva, as well as tissue, organ or cell type blood samples, blood lymphocyte samples and fractions thereof.

[0055] In a fourth aspect, the present invention relates to a method for producing a pharmaceutical composition, the method comprising the steps of identifying a candidate inhibitor or inhibitor as described herein and suitably formulating said candidate inhibitor or inhibitor into a pharmaceutical composition.

[0056] As used herein, the term "pharmaceutical composition" refers to a "suitable formulation" that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the composition may be administered. The pharmaceutical compositions of the present invention can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. To administer a binding compound according to the present invention by a particular route of administration, it may be necessary to coat the compound with or administer the compound together with a material that prevents the inactivation of the compound. For example, the compound may be administered to a subject in an appropriate carrier, such as liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0057] "Suitable carriers" refer to ingredients other than the active ingredient in a pharmaceutical formulation, which are non-toxic to the subject. Suitable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Administration can be, for example, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal (e.g., by injection or infusion). Prevention of the presence of microorganisms can be ensured both by sterilization procedures (as described above) and the use of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. Prolonged absorption of injectable dosage forms can also be achieved by the use of absorption delaying agents, such as aluminum monostearate and gelatin.

[0058] Regardless of the route of administration selected, the compound(s) of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention will be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may vary. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in the pharmaceutical arts.

[0059] The composition must be sterile and fluid, so long as it is deliverable by syringe. Isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride are often included in the composition.

[0060] The compositions of the present invention can be administered locally or systemically. Administration is generally parenteral, e.g., intravenously. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases.

[0061] In a fifth aspect, the present invention relates to an inhibitor as identified or a pharmaceutical composition as described herein for use in the prevention and / or treatment of an autoimmune disease in a subject, preferably while avoiding interference with the vasculoprotective function of EPCR.

[0062] As used herein, the terms "preventing" or "prevention" include administering the compound(s), preferably in a prophylactically effective amount, to a subject, with an eye toward reducing the subject's predisposition or risk, however slight, of developing an autoimmune disease, such as antiphospholipid syndrome, particularly primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis. In the case of prevention, the subject is preferably at risk of or susceptible to developing an autoimmune disease, such as antiphospholipid syndrome, particularly primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

[0063] The terms "treat" or "treatment", as used herein, include administering to the subject the compound(s), preferably in a therapeutically effective amount, to alleviate the disease or progression of an autoimmune disease, such as antiphospholipid syndrome, particularly primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

[0064] Preferred are embodiments of the invention wherein the inhibitor or pharmaceutical composition for use as described herein is selected from small molecules, peptides, antibodies or antigen-binding fragments thereof, enzymes, and aptamers.

[0065] Further preferred are embodiments in which the autoimmune disease is antiphospholipid syndrome (APS), in particular primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

[0066] In a sixth aspect, the present invention relates to a method for treating and / or preventing an autoimmune disease, such as antiphospholipid syndrome, in particular primary or secondary APS, primary Sjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, in a subject, comprising administering to said subject in need of such treatment and / or prevention an effective amount of an inhibitor as identified and described herein, or a pharmaceutical composition as described herein.

[0067] The term "administer" or "administration" as used herein includes enteral and topical administration, usually by injection, including, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0068] As used herein, an "effective amount" refers to an amount of a compound(s) or pharmaceutical composition(s) as described herein that normalizes the inflammatory state of a subject. This amount alleviates symptoms as seen in a disease and / or condition without being toxic to the subject. The dosing regimen will be determined by the attending physician and clinical factors. As is known in the medical arts, the dosage for any given patient will depend on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health, and other drugs being administered concomitantly. Typical doses can be, for example, in the range of 0.001 μg to 1000 μg (or amounts of nucleic acid for expression or inhibition of expression within this range). However, doses below or above this exemplary range are also contemplated, particularly considering the above factors.

[0069] The terms "of the invention," "in accordance with the invention," "according to the present invention," and the like, when used herein, are intended to refer to all aspects and embodiments of the present invention as described and / or claimed herein.

[0070] In the context of the present invention, the terms "about" and "approximately" refer to an interval of accuracy that a person skilled in the art would understand to be within which the technical effect of the feature in question remains certain. This term typically indicates a deviation from the specified numerical value of ±20%, ±15%, ±10%, e.g., ±5%. As will be understood by a person skilled in the art, the deviation from such a specific numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than a man-made or engineered technical effect. As will be understood by a person skilled in the art, the deviation from such a specific numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than a man-made or engineered technical effect. Where an indefinite or definite article, such as "a," "an," or "the," is used when referring to a singular noun, the plural of that noun is also included unless something specifically specified otherwise.

[0071] It should be understood that application of the teachings of the present invention to a particular problem or environment, and the inclusion of variations of the present invention or additional features thereof (such as further aspects and embodiments), are within the capabilities of those skilled in the art in view of the teachings contained herein.

[0072] All references, patents and publications cited herein are hereby incorporated by reference in their entirety. [Example]

[0073] Certain aspects and embodiments of the present invention will now be described, by way of example, with reference to the description, figures, and tables provided herein. Such examples of methods, uses, and other aspects of the present invention are merely representative and should not be considered to limit the scope of the present invention solely to such representative examples.

[0074] Example 1: EPCR-dependent signaling of aPL FXa generated by the clot initiator TF-FVIIa utilizes the endothelial cell protein C receptor (EPCR) for protease-activated receptor (PAR)2 cleavage, which is specifically required for LPS-induced interferon (IFN) responses (15, 16). Consistent with this pathway, an inhibitory antibody against EPCR (αEPCR 1560), but not a non-inhibitory antibody (αEPCR 1562), blocked LPS induction of interferon-regulated host defense genes (Figure 2A). Furthermore, proinflammatory TNFα was not induced in spleen-derived monocytes (Figure 1A). Unexpectedly, lipid-reactive IgG fractions from patients with active syphilis (Figure 1A) and well-characterized lipid-reactive monoclonal aPL with (HL5B) or without (HL7G) β2GPI cross-reactivity (Figure 1B) not only induced interferon-regulated genes but also induced EPCR-dependent TNFα. Although aPL promotes TNFα through amplification of Tlr7 signaling (9), the Tlr7 agonist R848 upregulated only TNFα, but not interferon-regulated genes (Fig. 1B), demonstrating that aPL engages EPCR in a novel pathway associated with host defense.

[0075] EPCR blockade similarly inhibited procoagulant and proinflammatory aPL responses in human monocytes (Figure 2B, Figure 2C). Function-blocking anti-mouse EPCR abolished widely established aPL monocyte responses (Figure 1D), namely, TF, Tnfa, and reactive oxygen species (ROS) production. These productions were independent of EPCR-protein C (PC) signaling (17) and Lrp8, a known coreceptor for β2GPI-dependent aPL pathogenesis (12, 13) (Figure 1D). Importantly, a novel mouse model of EPCR abolished the widely established aPL monocyte response (Figure 1D), namely, TF, Tnfa, and reactive oxygen species (ROS) production.C / S In mice, removal of the predicted intracellular palmitoylation acceptor Cys242 of EPCR by knock-in mutagenesis at Ser blocked aPL signaling, indicating that EPCR has a highly specific signaling function in aPL pathogenesis.

[0076] We analyzed IgG fractions from randomly selected patients representative of the diagnostic reactivity seen in the APS patient population (8, 11). Rare aPL IgG reactive with β2GPI alone (α-β2GPI; 2 / 20 patients) did not induce a rapid proinflammatory response, whereas lipid-reactive aPL IgG (defined by cardiolipin reactivity, α-CL) with or without β2GPI cross-reactivity (similar to monoclonal aPL HL7G; 7 / 20 patients) (similar to monoclonal aPL HL5B; 11 / 20 patients) significantly increased signaling activity in mice with EPCR. C / S In monocytes (Fig. 2D) or human trophoblasts, it was significantly reduced in the presence of inhibitory αEPCR (Fig. 2E). These data not only demonstrated that the majority of patient aPL had preserved lipid-responsive and EPCR-dependent signaling, but also demonstrated a remarkable interspecies conservation of this signaling mechanism in innate immune and germ cells.

[0077] The imaging revealed that aPL HL5B was EPCR deficient (EPCR low ) demonstrated that aPL did not bind to monocytes (18) or to cells blocked by inhibitory αEPCR 1560, whereas non-inhibitory αEPCR 1562 did not block binding or aPL internalization (Figure 1D). In contrast, aPL was blocked by EPCR C / STF bound to monocytes but was not internalized (Figure 1D). In human monocytes, aPL HL5B colocalized intracellularly with non-inhibitory αEPCR after 15 min of stimulation, whereas when EPCR was engaged with the Fab'2 fragment of the same aPL-deficient complement-binding protein, only surface binding was present and internalization was absent (Figure 1E). Complement is known to play a role in aPL pathogenesis (8, 19-21), inducing thiol-disulfide exchange and protein disulfide isomerase (PDI)-mediated conformational changes in TF. This increases TF coagulation activity (22) and allows coagulation-dependent TF-FVIIa transport in the ADP-ribosylation factor (ARF) 6 integrin pathway (23) to initiate aPL endosomal proinflammatory signaling (14). Inhibition of complement, PDI, and ARF6, as well as the coagulation proteases FXa and thrombin, not only blocked TF-FVIIa but also EPCR internalization (Fig. 1F), indicating that EPCR-bound aPL was internalized along with the TF-FVIIa complex, dependent on the cooperation of the innate immune defense complement and coagulation pathways.

[0078] Example 2: EPCR surface display of endosomal LBPA Although some aPLs interfere with anticoagulation (24), this feature was not shared by all lipid-reactive prototype aPLs (Fig. 3A). Among the anti-mouse EPCR antibodies that did not inhibit PC activation (Fig. 3A), a rare antibody, αEPCR 1682 (Fig. 2B), was identified that potently inhibited aPL proinflammatory signaling. Internalization without inhibiting aPL binding (Fig. 3C) indicates that αEPCR 1682 blocked a central pathway of aPL pathogenesis that is independent of clotting factors or the binding of aPL to EPCR.

[0079] Surprisingly, αEPCR 1682 was C / S EPCR, which was expressed at normal levels in monocytes from mice, did not stain (Fig. 3D). This was reasonable because EPCR interacts with FXa (15) and FXa is important for the formation and recycling of the TF pathway inhibitor (TFPI) complex (25).C / S Altered EPCR transport in mice prevented TF-FVIIa-FXa-TFPI complex formation and thus prevented the conformational changes required for αEPCR 1682 binding. Imaging of FXa bound to the surface of TFPI-deficient TfpiΔK1 monocytes (14) demonstrated that this complex indeed formed dependently on TFPI synthesized by the monocytes, and that EPCR 1682 binding was essential for the formation of FXa. C / S However, αEPCR 1682 stained TfpiΔK1 cells, except for the αEPCR 1682 reactivity required for FXa-EPCR interaction (Fig. 3D).

[0080] Because EPCR function depends on structurally bound lipids (26, 27), it was hypothesized that lipid exchange influences EPCR antibody reactivity. The late endosomal lipid (LBPA (lysobisphosphatidic acid, or bis(monoacylglycerol)phosphate (BMP)) is recognized by aPL after internalization (28), and EPCR and aPL are transported through a common endolysosomal compartment (Figure 1E). Supporting that LBPA replaces the structurally bound lipids of EPCR, non-permeabilized cells expressing EPCR stained with αLBPA 6C4, whereas EPCR-deficient cells also exhibited signaling-deficient EPCR. C / S The cells were also unstained (Fig. 3E).

[0081] Importantly, EPCR C / S The simple addition of LBPA to the culture medium of EPCR-deficient cells restored cell surface αLBPA 6C4 and αEPCR 1682 staining (Figure 3E) and promoted FXa surface localization (Figure 3D), but not EPCR-deficient cells. αEPCR 1682 also specifically blocked αLBPA 6C4 binding to mouse monocytes (Figure 3F). Conversely, αLBPA 6C4 competed with αEPCR 1682 binding, indicating that αEPCR 1682 recognized LBPA-loaded EPCR (Figure 3G). Notably, LBPA supplementation alone did not significantly inhibit EPCR. C / SSupplementation with the commonly putative aPL ligand cardiolipin (CL) or the negatively charged procoagulant phosphatidylserine (PS) restored cellular aPL proinflammatory signaling, but not with CL ( Fig. 3H ).

[0082] Exposure of purified human or mouse soluble EPCR (sEPCR) expressed in insect cells (15) to LBPA resulted in repurified proteins with a significant shift in mobility on native gels, demonstrating lipid exchange with LBPA (Figure 3I). Purified human sEPCR demonstrated tight binding of aPL HL5B to LBPA-loaded EPCR, whereas the binding affinity of unmodified sEPCR could not be quantified by surface plasmon resonance (SPR) (Figure 3J). Thus, EPCR-LBPA is the antigenic target recognized by aPL.

[0083] Competition experiments confirmed the high affinity of the aPL HL5B for LBPA-loaded sEPCR (Figures 4A and 4B), but LBPA loading did not increase the potency of EPCR to inhibit PC activation (Figure 4C). Only lipid-reactive aPL recognized LBPA-loaded mouse or human EPCR, but not β2GPI-specific aPL (Figures 4D and 4E). Cell binding assays (Figure 4F), competition experiments (Figure 4G), and monocyte activation readouts (Figure 4H) demonstrated somewhat higher affinity for the β2GPI-cross-reactive aPL HL7G compared with the lipid-selective aPL HL5B. Thus, the acquisition of protein reactivity during aPL evolution appears compatible with affinity maturation of the pathogenic target EPCR-LBPA. This finding may be important for interpreting the clinical correlation between β2GPI cross-reactivity and APS severity.

[0084] Example 3: EPCR-LBPA is a target for aPL-induced thrombosis It remained unclear why blocking surface lipid presentation by αEPCR-LBPA 1682 was sufficient to inhibit aPL signaling without blocking aPL binding. Because aPL rapidly induced the exposure of procoagulant phosphatidylserine (Fig. 4H), a process amplified by acid sphingomyelinase (ASM) (29), blocking ASM with desipramine indicated that ASM is required for aPL pathogenic signaling (Fig. 5A) and aPL internalization (Fig. 5B). Various agonists, including thrombin, induce ASM cell surface translocation (30-32). In human mononuclear cells, aPL stimulated maximal ASM activity within 15 min, dependent on FXa and thrombin-dependent PAR1 cleavage (Fig. 5C). However, ASM activity was not blocked by inhibitors of complement, PDI, or ARF6. This indicates that ASM activation required only coagulation activation, not TF-FVIIa internalization. This ASM activation pathway was conserved in mice (Fig. 6A). Importantly, aPL HL5B Fab'2 also induced ASM activity and promoted thrombin-dependent appearance of ASM on the cell surface (Fig. 5D). This confirms that ASM activation is an early event preceding aPL internalization and endosomal trafficking.

[0085] ASM requires LBPA for activity (33). ASM activation was not only blocked by an antibody that blocks the binding of aPL to EPCR, but also by αEPCR-LBPA 1682 (Fig. 6A). In a series of experiments, it was further demonstrated that EPCR-LBPA directly activated cell surface ASM. LBPA was bound to EPCR. C / SThe extracellular addition of αEPCR-LBPA restored ASM activation by aPL, but not by EPCR-deficient monocytes (Figure 6B). Thrombin stimulation, which induces ASM surface expression, was sufficient to trigger ASM activation, which was blocked by the extracellular addition of αEPCR-LBPA 1682 (Figure 6C). TfpiΔK1 cells expressed LBPA-loaded EPCR (Figure 3D) but lacked surface FXa, which triggers thrombin generation. ASM activation in these cells was not induced by aPL but was induced by thrombin in an EPCR-LBPA-dependent manner (Figure 6C). Addition of purified EPCR-LBPA to cell lysates from unstimulated cells also efficiently induced ASM activation, whereas unmodified EPCR did not, and this effect was specifically blocked by αEPCR-LBPA 1682 (Figure 5E). Thus, coagulation-induced PAR1 signaling translocates ASM due to cell surface activation by EPCR-LBPA. Consequently, ASM modification of the surface lipids is required for endosomal transport and signaling of EPCR-bound aPL.

[0086] Considering that monocytes mediate thrombosis (34), we first exploited the unique properties of the mouse monoclonal αEPCR 1650 and 1682. These murine monoclonal αEPCR 1650 and 1682 differentially regulated aPL pathogenic signaling while avoiding interference with the anticoagulant PC pathway (Figures 3A and 3B). Thrombosis was significantly attenuated by αEPCR-LBPA 1682 but not by the noninhibitory αEPCR 1650 (Figure 5F). Similarly, Lrp8-independent thrombosis induction by the dual-reactive aPL HL7G was specifically blocked by αEPCR-LBPA 1682 (Figures 5G and 5H).

[0087] Importantly, thrombosis induction by aPL HL5B was significantly reduced by EPCR when compared with lineage-matched WT controls. C / STo assess the broader implications of this finding for autoimmune pathology, we isolated IgG fractions from 16-week-old prothrombotic lupus-prone MRL-lpr mice (35) and age-matched lupus-free MRL control mice. Thrombosis induction by pathogenic IgG was significantly reduced by EPCR. C / S When injected into mice, this was reversed to the levels seen with IgG isolated from control mice (Fig. 5J), supporting the central role of the identified signaling targets for autoimmune disease-associated thrombosis.

[0088] Example 4: EPCR pathogenic signaling in fetal death The importance of this pathway in human trophoblast cells was assessed by knockdown of ALIX (Figure 8A). ALIX is required for normal liposome function. ALIX knockdown reduced cell surface display of LBPA but not EPCR expression (Figure 8B) and abolished aPL-induced, but not TNFα-induced, proinflammatory effects. However, supplementation with extracellular LBPA restored aPL signaling (Figure 7A). In support of a direct interaction between ASM and EPCR, proximity ligation assays (PLA) showed that EPCR and ASM colocalized after stimulation with thrombin or aPL Fab'2 HL5B (Figure 8C), but not in hirudin-treated cells without LBPA or ALIX. - / - This was not the case in cells (Fig. 7B). Furthermore, thrombin recruitment to ASM was also mediated by ALIX. - / - After LBPA exposure in cells, EPCR-LBPA showed increased proximity ligation with EPCR (Fig. 7C), thus EPCR-LBPA directly interacts with cell surface ASM and stimulates its activity.

[0089] Human ALIX - / - Trophoblast and mouse EPCR C / S Monocytes provided a means to compare the interspecies conservation of lipid presentation by EPCR. Only the addition of S / R 18:1 LBPA and R / R 18:1 LBPA significantly reduced the expression of aPL HL5B and ALIX. - / -Binding to trophoblast cells (Figure 7D) or EPCR C / S Restoring signaling in monocytes (Figure 7E), whereas S / S 18:1 LBPA or semi-S / R LBPA did not, human and mouse EPCR thus present LBPA with the same selectivity, explaining the extraordinary species cross-reactivity of pathogenic aPL.

[0090] Furthermore, we analyzed the role of EPCR in a mouse model of aPL-induced pregnancy loss. Although EPCR plays a central role in maintaining embryonic trophoblast function and survival (36), EPCR was significantly reduced compared to WT controls. C / S Mouse or EPCR low No significant embryonic loss was observed in mice (Figures 7F and 7G). However, EPCR signaling-deficient mice were protected from fetal death induced by the lipid-reactive aPL HL5B. These experiments demonstrate that the newly identified aPL-EPCR signaling pathway is crucial for the major pathology of APS, namely thrombosis and pregnancy loss induced by lipid-reactive aPL, as well as β2GPI-cross-reactive aPL in vivo (Figure 7H).

[0091] Example 5: Development of autoimmunity by aPL-induced interferon signaling Furthermore, we investigated whether the identified targets of lipid-responsive aPL contribute to the development of autoimmunity. Upregulation of interferon responses in circulating immune cells has been associated with the development of APS (38, 39). The induction of interferon-regulated genes (e.g., IRF8, GBP2, and GBP6) by lipid-responsive aPL was associated with EPCR. C / S Addition of LBPA restored the interferon response, as shown for GBP2, but not for LPS, in monocytes (Fig. 9A). Furthermore, IgG isolated from MRL / lpr lupus erythematosus mice, but not MRL control mice, induced an EPCR-LPBA-dependent interferon response in monocytes (Fig. 9B).

[0092] Coculture of human plasmacytoid dendritic cells (pDCs) with B cells in the presence of an agonist for Tlr7, which contributes to autoimmunity in lupus erythematosus (40, 41), required the addition of aPL to promote the production of cardiolipin-reactive antibodies (Figure 9C). Under these conditions, an antibody blocking EPCR function (αEPCR 1496) prevented the development of lipid-reactive antibodies, whereas a non-inhibitory antibody (αEPCR 1489) did not (Figure 9C). This suggests that EPCR-dependent interferon signaling drives the autoimmune antibody response.

[0093] In support of this conclusion, mouse pDCs were cultured using EPCR C / S Anticardiolipin antibody production was absent in B cells, but not in B cells, isolated from mice (Figure 9D). Addition of LBPA or interferon α inhibited the production of anticardiolipin antibodies in aPL signaling-deficient EPCR cells. C / S Coculture with pDCs restored the proliferation of anticardiolipin-producing B cells (Fig. 9D). In contrast, cells lacking LRP8, the β2GPI receptor, produced anticardiolipin antibodies normally in response to costimulation with aPL and a Tlr7 agonist (Fig. 9E). The appearance of lipid-reactive antibodies required type I IFN receptor expression by B cells, but not pDCs (Fig. 9F). This demonstrates that aPL induced pDC interferon production to stimulate B cell responses.

[0094] Therefore, we evaluated the development of aPL in an established model of APS. Immunization with lipid-reactive monoclonal or polyclonal antibodies induces the appearance of cardiolipin-reactive antibodies in mice (42, 43). Immunization with aPL HL5B, but not control IgG, induced robust anticardiolipin titers within 3–6 weeks, depending on Tlr7, whereas control IgG did not. - / -Mice showed a slightly enhanced response (Fig. 10A). Immunization with aPL induced the appearance of circulating B1 cells that reacted with labeled liposomes (44), and liposome staining was blocked by EPCR-LPBA but not by unmodified EPCR (Fig. 10B), indicating the proliferation of EPCR-LPBA-reactive B cells. Anticardiolipin titers were significantly higher in strain-matched WT control mice and LRP8 mice. - / - In sharp contrast to mice, immunized EPCR C / S This was not the case in mice (Fig. 10C). Thus, genetic disruption of EPCR signaling abolished the expansion of lipid-reactive antibodies induced by immunization with pathogenic human aPL.

[0095] APS can also be induced by immunization with human β2GPI (45), which is associated with EPCR WT Mice and EPCR C / S In mice, LBPA induced a similar high-titer IgG antibody response to human β2GPI (Fig. 10D). IgG titers against LBPA were measured by EPCR. WT This occurred only in mice immunized with EPCR, but not in those immunized with prothrombin (Fig. 10E). WT Only mouse-derived IgG induced TF activity and pro-inflammatory signaling in monocytes (Fig. 10F), indicating that EPCR is required for the development of autoimmunity in experimental APS.

[0096] Example 6: EPCR-LBPA signaling drives aPL expansion and autoimmune pathology in vivo Specific inhibition of EPCR-LBPA completely prevented the development of aPL (Fig. 11A) and double-stranded DNA autoantibodies, which were already detectable in 6-week-old MRL-Faslpr mice but not in control MRL / MpJ mice (Fig. 11B). Treatment of MRL-Faslpr mice with αEPCR-LBPA 1682 not only reduced the development of autoantibodies but also protected them from progressive renal pathology, as evidenced by reduced infiltration of CD3+ and F4 / 80+ immune cells in the kidney (Fig. 11C) and reduced renal pathology scores reflecting glomerular and interstitial damage (Fig. 11D).

[0097] In a separate experiment, MRL-Faslpr mice were treated with αEPCR-LBPA 1682 or αEPCR 1650 for 6 weeks and analyzed 2 weeks after the end of treatment. αEPCR-LBPA 1682 again specifically suppressed serum αLBPA and αCL titers to levels seen in age-matched MRL / MpJ control mice (Fig. 12A) and attenuated renal infiltration of CD45+ / F4 / 80+ immune cells as measured by flow cytometry (Fig. 12B). These infiltrating myeloid cells expressed IFN-γ (Fig. 12C). Albuminuria developed only in mice treated with noninhibitory αEPCR 1650, but not in mice treated with inhibitory αEPCR-LBPA 1682 or MRL / MpJ control mice (Fig. 12D). Thus, EPCR-LBPA signaling is important for both the development of lipid-reactive antibodies and renal autoimmune pathology in this endosomal TLR7-dependent animal model, and more generally, drives renal autoimmune pathology. [Explanation of symbols]

[0098] Figure 1A no inhibitor control IgG Syphilis IgG fold gene induction Figure 1B no inhibitor fold gene induction Figure 1C no inhibitor (fold induction) (0-15min) Figure 1E merged superposition Figure 1F EPCR internalization no inhibitor +Rivaroxaban +hirudin +hirudin +compstatin +PDI inhibitor +PDI inhibitor +ARF6 inhibitor +ARF6 inhibitor VIIa internalization TF internalization Figure 2A species name effect on aCL aPL effect on aCL aPL PC activation inhibition Mouse blocking non-blocking Human Figure 2B primary monocytes no inhibitor (fold induction) Figure 2C no inhibitor (fold induction) Figure 2D (fold induction) 1h 1 hour 3h 3 hours Figure 2E (fold induction) 1h 1 hour 3h 3 hours Figure 3A EPCR-dependent aPC generation (% of control) Figure 3B (fold induction) Figure 3D counts count number Figure 3F 6C4 surface binding 6C4 surface binding Figure 3G 1682 surface binding antibody concentration antibody concentration control Figure 3H (fold induction) Figure 3I Mouse Human Figure 3J Response Units Time (sx103) Time (seconds x 103) Figure 4A surface binding Figure 4B (fold induction) Figure 4C EPCR-dependent aPC generation (% of control) Figure 4D aPL binding aPL binding Figure 4E antibody concentration antibody concentration Figure 4F antibody concentration antibody concentration Figure 4H Annexin V aPL concentration aPL concentration Figure 5A HL5B+desipramine HL5B+desipramine HL7G+desipramine HL7G+desipramine desipramine (units / 106 cells) (Annexin V-FITC) (0-15min) (fold induction) Figure 5B no inhibitor +desipramine Figure 5C unst. Unstimulated no inhibitor +Rivaroxaban +hirudin +hirudin +compstatin +PDI inh. +PDI inhibitor +ARF6 inhibitor ASM activity ASM activity Figure 5D merged superposition +hirudin +hirudin Figure 5E no inhibitor ASMase Figure 5F Thrombus size no inhibitor Figure 5G Thrombus size no inhibitor Figure 5H Thrombus size Figure 5I Thrombus size Figure 5J Thrombus size Figure 6A unst. Unstimulated no inhibitor +Rivaroxaban +hirudin +hirudin +compstatin +PDI inh. +PDI inhibitor +ARF6 inhibitor ASMase Figure 6B ASMase Figure 6C thrombin no inhibitor ASMase Figure 7A (fold induction) scramble control Figure 7B PLA+ signals / cell PLA+ signals / cell Figure 7C PLA+ signals / cell PLA+ signals / cell thrombin thrombin+LBPA thrombin+LBPA Figure 7D merged superposition control JAR +semi LBPA +semi LBPA Figure 7E +semi LBPA +semi LBPA (fold induction Figure 7F HL5B immunized mice HL5B immunized mice naive mice Figure 7G control fetal death rate Figure 7H Endosomal Signaling Thrombosis Pregnancy loss Figure 8A Scrambled Control Figure 8B Fold increase in MFI (antibody / isotype control) Scrambled control Figure 8C PLA+ signals / cell PLA+ signals / cell Scrambled control hirudin thrombin Figure 9A (fold induction) Figure 9B control IgGs no inhibitor (fold induction) Figure 9C cardiolipin binding (OD units) unstimulated Figure 9D cardiolipin binding (OD units) B cells unstimulated Figure 9E cardiolipin binding (OD units) unstimulated Figure 9F cardiolipin binding (OD units) B cells unstimulated Figure 10A binding to cardiolipin (OD units) time (weeks) Figure 10B binding to cardiolipin (OD units) time (weeks) Figure 10C Count Count number PL vesicle+ cells PL vesicle+ cells Figure 10D hβ2GPI binding hβ2GPI binding (OD units) Figure 10E binding to indicated proteins (OD units) Figure 10F PCA (units / 106 cells) PCA (units / 106 cells) (fold induction) Figure 11C cardiolipin binding (OD units) time [days] time [days] Figure 11D ds DNA binding dsDNA binding (OD units) no inhibitor Figure 11E CD3+ cells / hpf CD3+ cells / hpf F4-80+ cells / hpf F4-80+ cells / hpf Figure 11F Score of renal pathology Figure 12A OD units Figure 12B Frequency (%) Frequency (%) Figure 12C Frequency (%) Frequency (%) Figure 12D Albumin Creatinine days after treatment start

[0099] References References cited herein are as follows: 1. B. Giannakopoulos, SA Krilis, The pathogenesis of the antiphospholipid syndrome. N Engl J Med 368, 1033-1044 (2013). 2. K. Schreiber et ah, Antiphospholipid syndrome. Nat Rev Dis Primers 4, 17103 (2018). 3. R. Bakimer et ah, Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H-3). J Clin Invest 89, 1558-1563 (1992). 4. S. S. Pierangeli, E. N. Harris, Induction of phospholipid-binding antibodies in mice and rabbits by immunization with human beta 2 glycoprotein 1 or anticardiolipin antibodies alone. Clin Exp Immunol 93, 269-272 (1993). 5. P. Lieby et al, The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity. Blood 97, 3820-3828 (2001). 6. P. Redecha et al., Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury. Blood 110, 2423-2431 (2007). 7. D. Manukyan et al., Cofactor-independent human antiphospholipid antibodies induce venous thrombosis in mice. J. Thromb. Haemost 14, 1011-1020 (2016). 8. N. Muller-Calleja et al., Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies. Blood Adv 2, 979-986 (2018). 9. N. Prinz et al, Antiphospholipid antibodies induce translocation of TLR7 and TLR8 to the endosome in human monocytes and plasmacytoid dendritic cells. Blood 118, 2322-2332 (2011). 10. N. Prinz, N. Clemens, A. Canisius, K. J. Lackner, Endosomal NADPH-oxidase is critical for induction of the tissue factor gene in monocytes and endothelial cells. Lessons from the antiphospholipid syndrome. Thromb. Haemost 109, 525-531 (2013). 11. N. Muller-Calleja et al., Antiphospholipid antibody-induced cellular responses depend on epitope specificity : implications for treatment of antiphospholipid syndrome. J. Thromb. Haemost 15, 2367-2376 (2017). 12. Z. Romay-Penabad et al., Apolipoprotein E receptor 2 is involved in the thrombotic complications in a murine model of the antiphospholipid syndrome. Blood 117, 1408-1414 (2011). 13. V. Ulrich et al, ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice. Arthritis Rheumatol 68, 730-739 (2016). 14. N. Miiller-Calleja et al., Tissue factor pathway inhibitor primes monocytes for antiphospholipid antibody-induced thrombosis. Blood, (2019). 15. J. Disse et al., The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors. J Biol. Chem 286, 5756-5767 (2011). 16. H. P. Liang et al, EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice. Blood 125, 2845-2854 (2015). 17. R. K. Sinha et al, Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization. Arterioscler. Thromb. Vase. Biol 36, 518-524 (2016). 18. E J. Castellino et al, Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge. Thromb. Haemost 88, 462-472 (2002). 19. S. S. Pierangeli et al, Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia. Arthritis Rheum 52, 2120-2124 (2005). 20. F. Fischetti et al, Thrombus formation induced by antibodies to beta2-glycoprotein I is complement dependent and requires a priming factor. Blood 106, 2340-2346 (2005). 21. G. Girardi et al., Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J. Clin. Invest 112, 1644-1654 (2003). 22. F. Langer et al., Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase. Blood 121, 2324-2335 (2013). 23. A. S. Rothmeier et al., Identification of the integrin-binding site on coagulation factor Vila required for proangiogenic PAR2 signaling. Blood 131, 674-685 (2018). 24. V. Hurtado et al., Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death. Blood 104, 1369-1374 (2004). 25. I. Ott et al, Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor. Arterioscler. Thromb. Vase. Biol 20, 874-882 (2000). 26. V. Oganesyan et al., The crystal structure of the endothelial protein C receptor and a bound phospholipid. J. Biol. Chem 277, 24851-24854 (2002). 27. J. Lopez-Sagaseta et al, sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove. Blood 119, 2914-2921 (2012). 28. T. Kobayashi et al, A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193-197 (1998). 29. J. Wang, U. R. Pendurthi, L. V. M. Rao, Sphingomyelin encrypts tissue factor: ATP- induced activation of A-SMase leads to tissue factor decryption and microvesicle shedding. Blood Adv 1, 849-862 (2017). 30. W. M. Kuebler et al., Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase. Am. J. Physiol Lung Cell Mol. Physiol 310, L720-L732 (2016). 31. P. Munzer et al., Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation. Arterioscler. Thromb. Vase. Biol 34, 61-71 (2014). 32. H. Grassme et al., CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem 276, 20589-20596 (2001). 33. V. O. Oninla, B. Breiden, J. O. Babalola, K. SandhofF, Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2. J. Lipid Res 55, 2606-2619 (2014). 34. S. Subramaniam et al, Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development. Blood 129, 2291-2302 (2017). 35. C. Moratz et al, Regulation of systemic tissue injury by coagulation inhibitors in B6.MRL / lpr autoimmune mice. Clin Immunol 197, 169-178 (2018). 36. B. Isermann et al., The thrombomodulin-protein C system is essential for the maintenance of pregnancy. Nat. Med 9, 331-337 (2003). 37. H. R Liang et al., Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice. Blood 126, 2415-2423 (2015). 38. C. Perez-Sanchez et al., Gene profiling reveals specific molecular pathways in the pathogenesis of atherosclerosis and cardiovascular disease in antiphospholipid syndrome, systemic lupus erythematosus and antiphospholipid syndrome with lupus. Ann. Rheum. Dis 74, 1441-1449 (2015). 39. E. Palli, E. Kravvariti, M. G. Tektonidou, Type I Interferon Signature in Primary Antiphospholipid Syndrome: Clinical and Laboratory Associations. Front Immunol 10, 487 (2019). 40. A. Sang et al, Innate and adaptive signals enhance differentiation and expansion of dual antibody autoreactive B cells in lupus. Nat Commun 9, 3973 (2018). 41. B. Giannakopoulos et al., Deletion of the antiphospholipid syndrome autoantigen beta2 - glycoprotein I potentiates the lupus autoimmune phenotype in a Toll-like receptor 7- mediated murine model. Arthritis Rheumatol 66, 2270-2280 (2014). 42. S. S. Pierangeli, E. N. Harris, Induction of phospholipid-binding antibodies in mice and rabbits by immunization with human beta 2 glycoprotein 1 or anticardiolipin antibodies alone. Clin Exp Immunol 93, 269-272 (1993). 43. R. Bakimer et al., Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H-3). J Clin Invest 89, 1558-1563 (1992). 44. P. Lieby et al, The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity. Blood 97, 3820-3828 (2001). 45. A. E. Gharavi, L. R. Sammaritano, J. Wen, K. B. Elkon, Induction of antiphospholipid autoantibodies by immunization with beta 2 glycoprotein I (apolipoprotein H). J Clin Invest 90, 1105-1109 (1992).

Claims

1. 1. A method for assisting in determining whether a subject is suffering from an autoimmune disease, comprising detecting binding between an antiphospholipid antibody (aPL) and lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or an LBPA-binding fragment thereof in a biological sample obtained from the subject, wherein the detection of binding between aPL and LBPA bound to endothelial cell protein C receptor (EPCR) or the LBPA-binding fragment thereof indicates an autoimmune disease in the subject, wherein the autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

2. 2. The method of claim 1, wherein the lysobisphosphatidic acid (LBPA) bound to endothelial cell protein C receptor (EPCR) or its LBPA-binding fragment is directly or indirectly immobilized on a solid support material.

3. 1. A method for identifying an inhibitor of endothelial cell protein C receptor (EPCR) function in an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, comprising: i) providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof; ii) contacting a candidate inhibitor with said sample; iii) testing the binding of LBPA to the EPCR protein or the LBPA-binding fragment thereof in the presence or absence of the inhibitor candidate; iv) identifying the inhibitor candidate as the inhibitor if the LBPA binding is absent when tested; A method comprising:

4. The method of claim 3 , wherein at least one of EPCR, LBPA, and / or the inhibitor candidate is labeled and / or immobilized.

5. 1. A method for identifying an inhibitor of endothelial cell protein C receptor (EPCR) function in an autoimmune disease, wherein the inhibitor does not interfere with the regulatory function of EPCR in blood coagulation, wherein the autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, comprising: i) providing a biological sample containing EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof; ii) binding LBPA to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA complex; iii) contacting a candidate inhibitor with the sample; iv) testing cellular function based on antiphospholipid antibody (aPL) binding or changes in protein expression of interferon-inducible genes in cells present in the biological sample in the presence or absence of the candidate inhibitor; v) identifying the candidate inhibitor as the inhibitor if the aPL binding is absent or the cellular function is absent when tested; A method comprising:

6. The method of claim 5 , wherein at least one of EPCR, LBPA, the inhibitor candidate, and / or aPL is labeled and / or immobilized.

7. 5. The method of claim 3 or 4, further comprising testing the identified candidate inhibitor as an inhibitor of endothelial cell protein C receptor (EPCR) function in an autoimmune disease selected from the group consisting of antiphospholipid syndrome (APS), primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

8. 7. The method of claim 5 or 6, further comprising testing the candidate inhibitor identified as an inhibitor of endothelial cell protein C receptor (EPCR) function in an autoimmune disease selected from the group consisting of antiphospholipid syndrome (APS), primary or secondary APS, primary Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, wherein the candidate inhibitor does not inhibit the regulatory function of EPCR in blood coagulation.

9. The method of any one of claims 3 to 8, wherein the inhibitor candidate is selected from a small molecule, a protein, a peptide, an antibody or an antigen-binding fragment thereof, an enzyme, and an aptamer.

10. 3. The method of claim 1 or 2, wherein the subject is a mammal or a human.

11. The method of any one of claims 3 to 9, wherein the biological sample is obtained from a subject, including a mammal or a human.

12. The biological sample is (1) Body fluids, including blood, serum, or saliva; (2) tissue, organ, or cell-type blood samples; (3) a sample of blood lymphocytes, or (4) Any one of the fractions (1) to (3) The method according to any one of claims 1 to 11, wherein the method is selected from the group consisting of:

13. 12. A method for producing a pharmaceutical composition, the method comprising the steps of identifying an inhibitor according to any one of claims 3 to 9 or 11, and formulating the inhibitor identified in said step into a pharmaceutical composition.

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