Abstract
The present invention relates to methods for detecting whether a subject suffers from an autoimmune disease, such as, for example, antiphospholipid syndrome (APS), by detecting antiphospholipid antibodies (aPL) in a sample using a novel target, the lysobisphosphatidic acid (LBPA) bound to the endothelial protein C receptor (EPCR) or an LBPA-binding fragment thereof. Furthermore, the present invention relates to methods for identifying an inhibitor of endothelial protein C receptor (EPCR) function in autoimmune disease, preferably without a side effect on EPCR regulatory function in coagulation, and a method for producing a pharmaceutical composition comprising the steps of identifying a potential inhibitor, and suitably formulating said potential inhibitor into a pharmaceutical composition. Moreover, the present invention relates to said inhibitor as identified or said pharmaceutical composition for use in the prevention and/or treatment of an autoimmune disease, such as, for example, an antiphospholipid syndrome, in a subject. Furthermore, the present invention relates to a method for treating and/or preventing an autoimmune disease, such as, for example, antiphospholipid syndrome, in a subject.
Claims
1. A method for detecting whether a subject suffers from an autoimmune disease, comprising detecting binding of antiphospholipid antibodies (aPL) in a biological sample obtained from said subject to lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or an LBPA-binding fragment thereof, wherein said binding of aPL to said lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or said LBPA-binding fragment thereof detects an autoimmune disease in said subject.
2. The method according to claim 1, wherein said autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
3. The method according to claim 1, wherein said lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or an LBPA-binding fragment thereof is immobilized directly or indirectly to a solid carrier material.
4. A method for identifying an inhibitor of endothelial protein C receptor (EPCR) function in an autoimmune disease, comprising i) providing a biological sample comprising an EPCR protein or an lysobisphosphatidic acid (LBPA)-binding fragment thereof, ii) contacting a potential inhibitor with said sample, iii) testing binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof in the presence or absence of said potential inhibitor, and iv) identifying said potential inhibitor based on said LBPA-binding as tested.
5. A method for identifying an inhibitor of endothelial protein C receptor (EPCR) function in autoimmune disease without interfering with EPCR regulatory function in coagulation, comprising: i) providing a biological sample comprising an EPCR protein or a lysobisphosphatidic acid (LBPA)-binding fragment thereof, ii) binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA-complex, iii) contacting a potential inhibitor with said sample, iv) testing binding of an antiphospholipid antibody (aPL) or cellular functions in the presence or absence of said potential inhibitor, and v) identifying said potential inhibitor based on interfering with said aPL-binding or cellular functions as tested.
6. The method according to claim 4, wherein at least one of EPCR, LBPA, said potential inhibitor and/or aPL is suitably labelled and/or immobilized.
7. The method according to claim 4, further comprising the step of testing said potential inhibitor as identified for being an inhibitor of endothelial protein C receptor (EPCR) function in autoimmune disease while not inhibiting regulatory functions of EPCR in coagulation.
8. The method according to claim 4, wherein said potential inhibitor is selected from a small molecule, a protein, a peptide, an antibody or antigen-binding fragment thereof, an enzyme, and an aptamer.
9. The method according to claim 1, wherein said subject is a human.
10. The method according to claim 1, wherein said biological sample is selected from blood, serum, saliva, a tissue, organ, cell, and a sample of blood lymphocytes.
11. A method for producing a pharmaceutical composition, comprising the steps of identifying a potential inhibitor or inhibitor according to claim 4, and suitably formulating said potential inhibitor or inhibitor into a pharmaceutical composition.
12-14. (canceled)
15. A method of treating and/or preventing an autoimmune disease, said method comprising administering to said subject in need of such treatment and/or prevention an effective amount of an inhibitor as identified according to claim 4.
16. The method according to claim 15, wherein said inhibitor is selected from a small molecule, a peptide, an antibody or antigen-binding fragment thereof, an enzyme, and an aptamer.
17. The method according to claim 15, wherein said autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
18. The method according to claim 5, further comprising the step of testing said potential inhibitor as identified for being an inhibitor of endothelial protein C receptor (EPCR) function in autoimmune disease while not inhibiting regulatory functions of EPCR in coagulation.
19. The method according to claim 5, wherein said potential inhibitor is selected from a small molecule, a protein, a peptide, an antibody or antigen-binding fragment thereof, an enzyme, and an aptamer.
20. A method of treating and/or preventing an autoimmune disease, said method comprising administering to said subject in need of such treatment and/or prevention an effective amount of an inhibitor as identified according to claim 5.
21. The method according to claim 20, wherein said inhibitor is selected from a small molecule, a peptide, an antibody or antigen-binding fragment thereof, an enzyme, and an aptamer.
22. The method according to claim 20, wherein said autoimmune disease is selected from the group consisting of antiphospholipid syndrome (APS), primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
23. A method for producing a pharmaceutical composition, comprising the steps of identifying a potential inhibitor or inhibitor according to claim 5, and suitably formulating said potential inhibitor or inhibitor into a pharmaceutical composition.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0070] The figures show:
[0071] FIG. 1: shows that EPCR is the receptor for aPL (A) EPCR-dependent induction of IFN-regulated genes in monocytes by LPS and IgG from patients infected with Treponema pallidum. (B) Induction of IFN-regulated genes by aPL. (C) TF and Tnfα mRNA induction by aPL HL5B or HL7G stimulation of CD115+ splenocytes of indicated mice stimulated for 3 hours, as well as early ROS production; mean±SD, n=6; * p<0.0001; one-way ANOVA, Dunnett multiple-comparison test. (D) Live cell imaging of HL5B internalization in monocytes of indicated mouse strains. Bar=5 μm. (E) Live cell imaging of aPL HL5B Fab′2 or IgG colocalization with EPCR using non-inhibitory αEPCR 1489 in human MM1 cells. (F) Internalization of EPCR, FVIIa, and TF in MM1 cells stimulated for 15 minutes similarly required proteases and integrin trafficking. For quantification of internalization, surface staining was quenched with 0.4% trypan blue; mean±SD, n=6. * p<0.0001; one-way ANOVA, Dunnett multiple-comparison test compared to IgG control.
[0072] FIG. 2: shows that EPCR is required for aPL signaling. (A) Overview of functional properties of αEPCR against human and mouse EPCR. (B, C) TNF and TF induction in primary monocytes (B) and MM1 cells (C) stimulated for 3 h with HL5B or HL7G and pretreated for 15 minutes with anti-human EPCR antibodies; mean±SD, n=6. (D) CD115+ splenocytes of indicated mouse strains and (E) trophoblast cell induction of TNFα after 1 or 3 hours of stimulation with IgG isolated from APS patients (100 μg/ml) demonstrating cardiolipin reactivity alone (αCL), αβ2GP reactivity alone or dual reactivity. Human trophoblast cells were pretreated with either non-inhibitory αEPCR 1489 or inhibitory αEPCR 1496.
[0073] FIG. 3: shows that EPCR presents late endosomal lysobisphosphatidic acid (LBPA) on the cell surface. (A) Effect of aPL HL5B, aPL HL7G, and αEPCR antibodies on EPCR-dependent aPC generation on murine 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 multiple-comparison. (C) Effect of αEPCR on aPL HL5B and HL7G internalization in CD115+ splenocytes. Bar=5 μm. (D) Flow cytometry detection of FXa and EPCR on CD115+ spleen monocytes isolated from indicated mouse strains. (E) Effect of pre-treatment with 10 μM LBPA for 10 min on surface binding of αEPCR 1682 and αLBPA 6C4 on indicated monocytes; mean±SD, n=6. * p<0.003; multiple t-tests. (F) Competition of αEPCR 1650 and 1682 with binding of FITC-labelled anti-LBPA antibody 6C4 to mouse CD115+ splenocytes. (G) Competition of αLBPA 6C4 with binding of αEPCR 1682 to mouse monocytes. (H) Effect of LBPA, cardiolipin (CL), and phosphatidylserine (PS) (10 μM) on aPL HLSB signalling in EPCR.sup.C/S monocytes. Induction of TNF after 3 hours is shown; mean±SD, n=6. (I) LBPA loading of purified mouse or human sEPCR evidenced by faster mobility on native gels. (J) Surface plasmon resonance analysis of aPL HLSB binding to purified human sEPCR or sEPCR-LBPA. The affinity calculation was based on a monovalent binding model because no cooperative binding was evident.
[0074] FIG. 4: shows the effect of EPCR LBPA loading on aPL interaction. (A) Competition by sEPCR either loaded with LBPA or unmodified with binding of FITC-labeled HLSB Fab′2 fragments or control to mouse monocytes by flow cytometry. (B) LBPA-loaded EPCR is a more potent inhibitor than unmodified EPCR in blocking aPL HLSB signaling. (C) LBPA loading of human sEPCR does not alter competition of sEPCR with aPC generation on mouse endothelial cells. (D) Binding of HLSB to CHO cell control and CHO cells expressing mouse EPCR (mEPCR). Cells were either untreated or pre-incubated for 30 min with 10 μM LBPA, mean±SD, n=6. (E) Binding of anti-β2GPI aPL rJGG9 or control IgG to moue EPCR transfected CHO measured in a fluorescence microplate reader, mean±SD, n=3. (F) Binding of aPL HLSB, aPL HL7G or control IgG to mouse (mEPCR) or human (hEPCR) transfected CHO cells. Cells were loaded with 10 μM LBPA for 30 minutes before staining. (G) Binding of aPL HLSB (left panel) or HL7G (right panel) to LBPA-loaded mouse EPCR after preincubation for 15 minutes with different concentrations of purified sEPCR either unmodified or loaded with LBPA; mean±SD, n=6. (H) Dose response curve of HLSB and HL7G triggered PS exposure measured by annexin 5 surface staining.
[0075] FIG. 5: shows that aPL promote EPCR-LBPA activation of cell surface acidic sphingomyelinase and thrombosis. (A) aPL-mediated TF activation, PS exposure measured by annexin 5 staining, ROS production and TNFα induction as well as (B) aPL internalization in MM1 cells was blocked by sphingomyelinase inhibitor desipramine. Bar=5 μm. (C) aPL-induced ASM activity in MM1 cells is blocked by inhibitors of FXa, thrombin, and PAR1 cleavage. (D) Live cell imaging of surface ASM exposure in MM1 cells after 30 minutes of stimulation with Fab′2 aPL HLSB. Bar=5 μm. (E) ASM activity in unstimulated cell lysates after addition of sEPCR-LBPA (2.504) is blocked by αEPCR 1682. For all ASM activity assays: mean±SD, n=3. * p<0.0003; one-way ANOVA, Dunnett multiple-comparison test. (F) HLSB-induced thrombosis analyzed in the flow restricted vena cava inferior of WT mice treated with the indicated αEPCR antibodies. (G, H) Thrombosis induction by dual reactive aPL HL7G in the indicated mouse strains or WT mice in presence of indicated αEPCR. (F-H) Quantification of thrombus size 3 hours after aPL injection; median, interquartile range, and range; n=6-11; * p <0.004; one-way ANOVA, Dunnett multiple-comparison test compared to αEPCR 1650. (I, J) Thrombosis induction by aPL HLSB (I) or IgG isolated from age-matched 16 weeks old lupus-prone MRL/lpr and MRL control mice (J) in the indicated mouse strains. Quantification of thrombus size 3 hours after aPL injection; 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 comparisons test.
[0076] FIG. 6: shows that aPL promote EPCR-LBPA activation of cell surface acidic sphingomyelinase. (A) WT CD115+ spleen monocyte induction of ASM activity after 15 minutes aPL HLSB stimulation with the indicated inhibitors. (B) LBPA (10 μM) loading of EPCR.sup.C/S cells enabled ASM activation in CD115+ monocytes stimulated with HLSB. (C) aPL HLSB did not activate ASM in TfpiΔK1 cells, but thrombin (1 U/ml) activation of ASM in WT and TfpiΔK1 cells was blocked by αEPCR 1682, but not αEPCR 1650.
[0077] FIG. 7: shows that aPL-EPCR signalling promotes foetal loss. (A) TNFα mRNA induction after 2 hours by HLSB is prevented in Alix-deficient trophoblast cells; mean±SD, n=6. * p<0.0001; t-test following Shapiro-Wilk test for normal distribution. (B, C) Proximity ligation assays (PLA) for ASM and EPCR on scrambled control JARs or ALIX−/− cells after 10 minutes of stimulation with HLSB (B) or thrombin (C) with or without LBPA loading. Bar=25 μm. (D) aPL internalization in ALIX deficient JAR cells and signalling in EPCR.sup.C/S monocytes (E) was restored by adding 10 μM LBPA (S,R) but not by other phospholipids. (F) Pregnancy loss was scored at day 15.5 p.c. after injection of aPL HLSB on days 8 and 12. * p<0.02; one-way ANOVA, Dunnett multiple-comparison test. (G) Schematic representation of aPL signalling leading to thrombosis or pregnancy complications.
[0078] FIG. 8: shows that EPCR-LPBA is required for aPL signalling in trophoblast cells. (A) WB analysis of ALIX deficient JAR cells. (B) Loss of LBPA surface expression in ALIX knockdown trophoblast (JAR) cells expressing EPCR. Cells were stained with FITC labelled αEPCR or αLBPA antibodies and antibody surface binding was detected using a microplate fluorometer. (C) Proximity ligation assays (PLA) for ASM and EPCR on scrambled control JAR cells after 10 minutes of stimulation with thrombin and HL5B with or without thrombon inhibitor hirudin. Bar=25 μm.
[0079] FIG. 9: shows that EPCR is required for aPL interferon signalling and the expansion of B cells producing lipid-reactive aPL. (A) Gbp2 mRNA induction after 1 hour stimulation with HL5B, HL7G, or LPS (100 ng/ml) in EPCR.sup.C/S or WT monocytes with or without addition of LBPA. (B) WT monocytes were stimulated for 1 hour with IgG isolated from MRL/lpr lupus-prone or control MRL mice in the presence of the indicated antibodies to EPCR. (C) Human monocyte-derived DC were co-cultured with B cells in the presence of TLR7/8 agonist R848 and aPL HL5B with the indicated antibodies to human EPCR. Anti-cardiolipin titers were determined after 10 days. (D-F) Co-cultures of isolated spleen plasmacytoid dendritic cells (pDC) and B cells from the indicated mouse strains were co-cultured with Tlr7 agonist R848 and aPL HL5B for 10 days, followed by determination of anti-cardiolipin titers. IFNR.sup.−/−, type I interferon receptor deficient mice.
[0080] FIG. 10: shows that EPCR signalling drives aPL expansion in vivo. (A, B) Mice of the indicated genotypes were immunized with aPL HL5B or isotype matched control IgG and serum anti-cardiolipin titers were determined at the indicated times. (C) Cell reactive with negatively charged liposomes were only detected in mice immunized with aPL HL5B, but not isotype matched IgG. EPCR-LBPA, but not EPCR competed with liposome binding to these CD19+CD5+CD43+CD27+ memory-type B1a cells(D) Immunization with human β2GPI induced a similar high titer IgG antibody response to human β2GPI in EPCR.sup.WT and EPCR.sup.C/S mice. (E) Antibody titers to LBPA, but not mouse prothrombin, were only detected in EPCR.sup.WT, but not in EPCR.sup.C/S mice after 5 immunizations with human β2GPI. (F) IgG from human β2GPI-immunized EPCR.sup.WT, but not EPCR.sup.C/S mice induced monocyte TF activity and proinflammatory signalling in monocytes.
[0081] FIG. 11: shows the therapeutic relevance of an intervention in the EPCR-LBPA pathway in the exemplary context of autoimmunity and lupus erythematosus. (A) MRL-Faslpr lupus-prone mice were treated with the indicated αEPCR antibodies at an age of 4 weeks (day 0) and anti-cardiolipin titers were determined in serum at the indicated time points; n=5, *P=0.03; **P<0.0001; two-way ANOVA, Sidak's multiple comparisons test. (B) Antibodies to double stranded (ds) DNA were measured in αEPCR 1650- and αEPCR-LBPA 1682-treated MRL-Faslpr mice 2 weeks after the last dose or in 6-week-old MRL/MpJ control or MRL-Faslpr mice; n=4-5, *P<0.0001. (C) Immune cell infiltration of αEPCR-treated MRL-Faslpr mice; n=5, *P<0.025. (D) Renal pathology scores of αEPCR-treated MRL-Faslpr mice; n=5, *P=0.0317; Mann-Whitney U test.
[0082] FIG. 12: shows that EPCR-LBPA is required for the development of autoimmune disease. (A) Reactivity of purified IgG (40 μg/ml) from MRL/MpJ control mice and from MRL-Faslpr mice treated with αEPCR 1650 or αEPCR-LBPA 1682 with immobilized LBPA or cardiolipin; n=6-7, * P<0.0001, different from control αEPCR 1650 treated mice; two-way ANOVA, Sidak's multiple comparisons test. (B) Infiltration of kidneys with CD45+/F4/80+ immune cells in MRL-Faslpr mice treated with non-inhibitory αEPCR 1650 or inhibitory αEPCR-LBPA 1682; n=5-7, *P=0.024. (C) Phenotype of F4/80+ cells in kidneys of MRL-Faslpr mice determined by cytokine staining. (D) Albuminuria in MRL/MpJ control mice and in MRL-Faslpr mice treated at the age of four weeks for six weeks with the indicated antibodies.
EXAMPLES
[0083] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.
Example 1: EPCR-Dependent Signaling of aPL
[0084] FXa generated by the coagulation initiator TF-FVIIa utilizes the endothelial protein C receptor (EPCR) for protease activated receptor (PAR) 2 cleavage that is specifically required for LPS-induced interferon (IFN) responses (15, 16). In accord with this pathway, inhibitory (αEPCR 1560), but not non-inhibitory (αEPCR 1562) antibodies to EPCR (FIG. 2A) blocked LPS induction of interferon-regulated host defense genes, but not the induction of pro-inflammatory TNFα in spleen-derived monocytes (FIG. 1A). Unexpectedly, lipid-reactive IgG fractions from patients with active syphilis (FIG. 1A) and well characterized lipid-reactive monoclonal aPL without (HLSB) or with (HL7G) β2GPI cross-reactivity (FIG. 1B) not only induced interferon-regulated genes, but also TNFα dependent on EPCR. Although aPL promote TNFα through amplification of Tlr7 signaling (9), the Tlr7 agonist R848 upregulated only TNFα, but not interferon-regulated genes (FIG. 1B), demonstrating that aPL engage EPCR in a novel pathway related to host defense.
[0085] EPCR blockade similarly inhibited procoagulant and proinflammatory aPL responses in human monocytes (FIG. 2B, C). Function-blocking anti-mouse EPCR abolished broadly established aPL monocyte responses (FIG. 1D), i.e. TF, Tnfα and reactive oxygen species (ROS) production, that were independent of Lrp8 (FIG. 1D), a known co-receptor for EPCR-protein C (PC) signaling (17) and β2GPI-dependent aPL pathogenesis (12, 13). Importantly, elimination of the predicted EPCR intracellular palmitoylation acceptor Cys242 by knock-in mutagenesis to Ser in a novel mouse model, EPCR.sup.C/S mice, prevented aPL signaling, indicating that EPCR has a highly specific signaling function in aPL pathology.
[0086] Randomly selected patient IgG fractions representative of diagnostic reactivities found in general patient populations with APS (8, 11) were analyzed. Rare aPL IgG reactive with β2GPI alone (α-β2GPI; 2/20 patients) did not induce rapid proinflammatory responses, but signaling of lipid-reactive aPL IgG (defined by cardiolipin reactivity, a-CL) with (similar to monoclonal aPL HL7G; 7/20 patients) or without (similar to monoclonal aPL HL5B; 11/20 patients) β2GPI cross-reactivity was markedly reduced on mouse EPCR.sup.C/S monocytes (FIG. 2D) or human trophoblast cells in the presence of inhibitory αEPCR (FIG. 2E). These data showed not only that the vast majority of patient aPL preserved lipid-reactivity and EPCR-dependent signaling, but also a remarkable species preservation of this signaling mechanism in innate immune and embryonic cells.
[0087] Imaging demonstrated that aPL HL5B did not bind to EPCR-deficient (EPCR.sup.low) monocytes (18) or cells blocked by the inhibitory αEPCR 1560, whereas the non-inhibitory αEPCR 1562 prevented neither binding nor aPL internalization (FIG. 1D). In contrast, aPL bound to EPCR.sup.C/S monocytes, but did not internalize (FIG. 1D). On human monocytes, aPL HL5B colocalized intracellularly with a non-inhibitory αEPCR after 15 minutes of stimulation, but there was only surface binding and no internalization when EPCR was engaged by Fab′2 fragments of the same aPL lacking complement-fixation (FIG. 1E). Complement is a known player in aPL pathologies (8, 19-21) and causes thiol-disulfide exchange and protein disulfide isomerase (PDI) mediated conformational changes in TF. This increases TF clotting activity (22) and enables coagulation-dependent TF-FVIIa trafficking in the ADP-ribosylation factor (ARF) 6 integrin pathway (23) to initiated aPL endosomal proinflammatory signaling (14). Inhibition of complement, PDI, and ARF6, as well as coagulation proteases FXa and thrombin, prevented not only TF-FVIIa, but also EPCR internalization (FIG. 1F), indicating that EPCR-bound aPL internalized together with the TF-FVIIa complex dependent on a cooperation of innate immune defense complement and coagulation pathways.
Example 2: EPCR Surface Presentation of Endosomal LBPA
[0088] Certain aPL interfere with anticoagulation (24), but this feature was not common to all lipid-reactive prototypic aPL (FIG. 3A). Among anti-mouse EPCR antibodies that did not inhibit PC activation (FIG. 3A), a rare antibody, αEPCR 1682, with potent inhibition of aPL pro-inflammatory signaling (FIG. 2B) was identified and internalization without inhibiting aPL binding (FIG. 3C), indicating that αEPCR 1682 blocked a central pathway of aPL pathogenesis unrelated to coagulation factor or aPL binding to EPCR.
[0089] αEPCR 1682 surprisingly did not stain EPCR that was expressed at normal levels on monocytes from EPCR.sup.C/S mice (FIG. 3D). Since EPCR interacts with FXa (15) and FXa is crucial for TF pathway inhibitor (TFPI) complex formation and recycling (25), this was justified because altered EPCR trafficking in EPCR.sup.C/S mice prevented TF-FVIIa-FXa-TFPI complex formation and thus conformational changes required for αEPCR 1682 binding. Imaging surface bound FXa on TFPI-deficient TfpiΔK1 monocytes (14) showed that this complex indeed formed dependent on monocyte-synthesized TFPI and was absent in EPCR.sup.C/S cells. However, αEPCR 1682 stained TfpiΔK1 cells, excluding that αEPCR 1682 reactivity required FXa-EPCR interaction (FIG. 3D).
[0090] Because EPCR function is dependent on structurally bound lipid (26, 27), it was hypothesized that lipid exchange influenced 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 trafficked through a common endo-lysosomal compartment (FIG. 1E). Supporting the possibility that LBPA replaced the structurally bound lipid of EPCR, non-permeabilized cells that express EPCR, but not EPCR-deficient or signaling-defective EPCR.sup.C/S cells, were stained with αLBPA 6C4 (FIG. 3E).
[0091] Importantly, simply adding LBPA to the culture medium of EPCR.sup.C/S, but not EPCR-deficient cells restored cell surface αLBPA 6C4 and αEPCR 1682 staining (FIG. 3E) and promoted FXa surface localization (FIG. 3D). In addition, αEPCR 1682 specifically prevented binding of αLBPA 6C4 to mouse monocytes (FIG. 3F). Conversely, competition of αLBPA 6C4 with αEPCR 1682 binding showed that αEPCR 1682 recognized LBPA-loaded EPCR (FIG. 3G). Remarkably, only supplementation with LBPA, but not with the commonly assumed aPL ligand cardiolipin (CL) or the negatively charged procoagulant phosphatidylserine (PS) restored aPL pro-inflammatory signaling of EPCR.sup.C/S cells (FIG. 3H).
[0092] Exposure of purified insect cell-expressed human or mouse soluble EPCR (sEPCR) (15) to LPBA yielded a re-purified protein with a marked shift in mobility on native gels, demonstrating lipid exchange with LBPA (FIG. 3I). Purified human sEPCR showed tight binding of aPL HL5B with LBPA-loaded EPCR, whereas binding affinity could not be quantified by surface plasmon resonance with unmodified sEPCR (FIG. 3J). Thus, EPCR-LBPA is the antigenic target recognized by aPL.
[0093] Competition experiments confirmed the high affinity of aPL HL5B for LBPA-loaded sEPCR (FIG. 4A, B), while LBPA loading did not increase the potency of EPCR to inhibit PC activation (FIG. 4C). Only lipid-reactive, but not β2GPI-specific aPL recognized mouse or human EPCR loaded with LBPA (FIG. 4D-E). Cellular binding assays (FIG. 4F), competition experiments (FIG. 4G) and a monocyte activation readout (FIG. 4H) indicated a somewhat higher affinity of β2GPI cross-reactive aPL HL7G in comparison to lipid-selective aPL HLSB. Thus, acquisition of protein-reactivity during evolution of aPL appears to be compatible with affinity maturation for the pathogenic target EPCR-LBPA; this finding may be of importance for interpreting clinical correlations of β2GPI cross-reactivity with APS severity.
Example 3: EPCR-LBPA is the Target for aPL-Induced Thrombosis
[0094] It remained unclear why blockade of surface lipid-presentation by αEPCR-LBPA 1682 was sufficient to inhibit aPL signaling without preventing aPL binding. Because aPL rapidly induced procoagulant phosphatidylserine exposure (FIG. 4H), a process amplified by acidic sphingomyelinase (ASM) (29), ASM was blocked with desipramine and ASM was considered necessary for aPL pathogenic signaling (FIG. 5A) and aPL internalization (FIG. 5B). Various agonists, including thrombin, induce ASM cell surface translocation (30-32). Within 15 minutes, aPL maximally stimulated ASM activity in human monocytic cells dependent on FXa and thrombin-dependent PAR1 cleavage (FIG. 5C). However, ASM activity was not blocked by inhibitors of complement, PDI, or ARF6, indicating that ASM activation solely required coagulation activation, but not TF-FVIIa internalization. This pathway of ASM activation was conserved in the mouse (FIG. 6A). Importantly, Fab′2 of aPL HLSB also induced ASM activity and promoted thrombin-dependent appearance of ASM on the cell surface (FIG. 5D), confirming that ASM activation is an early event that precedes aPL internalization and endosomal trafficking.
[0095] ASM requires LBPA for activity (33). ASM activation was not only prevented by antibodies preventing aPL binding to EPCR, but also by αEPCR-LBPA 1682 (FIG. 6A). In a series of experiments, it was further showed that EPCR-LBPA directly activated cell surface ASM. Extracellular addition of LBPA to EPCR.sup.C/S but not to EPCR-deficient monocytes restored ASM activation by aPL (FIG. 6B). Thrombin stimulation to induce ASM surface expression was sufficient to trigger ASM activation that was blocked by extracellular addition of αEPCR-LBPA 1682 (FIG. 6C). TfpiΔK1 cells expressed LBPA-loaded EPCR (FIG. 3D), but lack surface FXa to trigger thrombin generation. ASM activation in these cells was not induced by aPL, but by thrombin in dependence of EPCR-LBPA (FIG. 6C). Addition of purified EPCR-LBPA, but not unmodified EPCR to cell lysates of unstimulated cells also efficiently induced ASM activity and this effect was blocked specifically by αEPCR-LBPA 1682 (FIG. 5E). Thus, coagulation-induced PAR1 signaling translocates ASM for cell surface activation by EPCR-LBPA. In turn, ASM modification of surface lipid modification is required for endosomal trafficking and signaling of EPCR-bound aPL.
[0096] Given that monocytes cause thrombosis (34), first the unique properties of mouse monoclonal αEPCR 1650 and 1682 were exploited, which lacked interference with the anti-coagulant PC pathway, while differentially regulating aPL pathogenic signaling (FIG. 3A, B). Thrombosis was markedly attenuated by αEPCR-LBPA 1682, but not by the non-inhibitory αEPCR 1650 (FIG. 5F). Similarly, Lrp8-independent thrombosis induction by the dual-reactive aPL HL7G was blocked specifically by αEPCR-LBPA 1682 (FIG. 5G, H).
[0097] Importantly, thrombosis induction by aPL HL5B was markedly reduced in EPCR.sup.C/S as compared to strain-matched WT controls (FIG. 51). In order to assess the broader implications of these finding for autoimmune pathologies, IgG fractions from 16 weeks old prothrombotic lupus-prone MRL-lpr mice (35) and age-matched lupus-free MRL control mice were isolated. Thrombosis induction by pathogenic IgG was reversed when injected into EPCR.sup.C/S mice to levels seen with IgG isolated from control mice (FIG. 5J), confirming the central role of the identified signaling target for thrombosis associated with autoimmune disease.
Example 4: EPCR Pathogenic Signaling in Fetal Loss
[0098] The importance of this pathway in human trophoblast cells by knock-down of ALIX (FIG. 8A) was evaluated, which is required for normal lysosomal functioning. ALIX knock-down diminished LBPA cell surface presentation, but not EPCR expression (FIG. 8B) and abolished aPL-induced, but not TNFα-induced proinflammatory effects. However, supplementing extracellular LBPA restored aPL signaling (FIG. 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 HLSB (FIG. 8C) but not in hirudin-treated or ALIX.sup.−/− cells without additional of LBPA (FIG. 7B). In addition, thrombin recruitment of ASM showed increased proximity ligation with EPCR in ALIX.sup.−/− cells after exposure to LBPA (FIG. 7C). Thus, EPCR-LBPA directly interacts with cell surface ASM to stimulate its activity.
[0099] Human ALIX.sup.−/− trophoblast cells and murine EPCR.sup.C/S monocytes provided tools to compare the species conservation of lipid presentation by EPCR. Only addition of S/R 18:1 LBPA and R/R 18:1 LBPA, but not S/S 18:1 LBPA or semi-S/R LBPA restored aPL HL5B binding to ALIX.sup.−/− trophoblast cells (FIG. 7D) or signaling in EPCR.sup.C/S monocytes (FIG. 7E). Thus, human and mouse EPCR present LBPA with the same selectivity, providing an explanation for the remarkable species cross-reactivity of pathogenic aPL.
[0100] Further, the role of EPCR in a mouse model of aPL-induced pregnancy loss was analyzed. Although EPCR plays a pivotal role in maintaining embryonic trophoblast function and survival (36), no significant embryo loss in EPCR.sup.C/S mice or EPCR.sup.low mice relative to WT controls (FIG. 7F, G) was found. However, EPCR signaling-deficient mice were protected from fetal loss induced by lipid-reactive aPL HL5B. These experiments show that the newly identified aPL-EPCR signaling pathway is crucial for the major pathologies of APS, i.e. thrombosis and pregnancy loss, induced by lipid-reactive, as well as β2GPI-cross-reactive aPL in vivo (FIG. 7H).
Example 5: Development of Autoimmunity by aPL-Induced Interferon Signalling
[0101] Further, it was investigated whether the identified target for lipid-reactive aPL contributes to the development of autoimmunity. Upregulation of interferon responses in circulating immune cells are linked to the development of APS (38, 39). Induction of interferon-regulated genes (e.g. IRF8, GBP2, GBP6) by lipid-reactive aPL, but not by LPS, was abolished in EPCR.sup.C/S monocytes and, as shown for GBP2, LBPA addition restored interferon responses (FIG. 9A). In addition, IgG isolated from MRL/lpr lupus erythematosus mice, but not MRL control mice induced interferon responses dependent on EPCR-LPBA in monocytes (FIG. 9B).
[0102] Co-cultures of human plasmacytoid dendritic cells (pDC) with B cells in the presence of an agonist for Tlr7, which contributes to auto-immunity in lupus erythematosus (40, 41), required addition of aPL to promote the production of cardiolipin-reactive antibodies (FIG. 9C). Under these conditions, a function-blocking (αEPCR1496), but not non-inhibitory (αEPCR1489) antibody to EPCR prevented the development of lipid-reactive antibodies (FIG. 9C), suggesting that EPCR-dependent interferon signaling drives autoimmune antibody responses.
[0103] Supporting this conclusion, anti-cardiolipin antibody production was absent when mouse pDC, but not B cells, were isolated from EPCR.sup.C/S mice (FIG. 9D). Addition of LBPA or interferon a restored the expansion of anti-cardiolipin producing B cells in co-cultures with aPL signaling-deficient EPCR.sup.C/S pDC (FIG. 9D). In contrast, cells deficient in LRP8, the receptor for β2GPI, produced anti-cardiolipin antibodies normally in response to co-stimulation of aPL and Tlr7 agonist (FIG. 9E). Appearance of lipid-reactive antibodies required type I IFN receptor expression by B cells, but not pDC (FIG. 9F), demonstrating that aPL induced pDC interferon production to stimulate B cell responses.
[0104] Therefore, the development of aPL in established models of APS was evaluated. 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 anti-cardiolipin titers within 3-6 weeks dependent on Tlr7, whereas Tlr9.sup.−/− mice displayed a slightly enhanced response (FIG. 10A). Immunization with aPL induced the appearance of circulating B1 cells reactive with labeled liposomes (44) and liposome staining was prevented by EPCR-LPBA, but not unmodified EPCR (FIG. 10B), indicating the expansion of EPCR-LPBA reactive B cells. Anti-cardiolipin titers did not develop in immunized EPCR.sup.C/S mice in sharp contrast to strain-matched WT controls as well as LRP8.sup.−/− mice (FIG. 10C). Thus, genetic ablation of EPCR signaling abolished the expansion of lipid-reactive antibodies triggered by immunization by pathogenic human aPL.
[0105] APS is also triggered by immunization with human β2GPI (45) which induced a similar high titer IgG antibody response to human β2GPI in EPCR.sup.WT and EPCR.sup.C/S mice (FIG. 10D). IgG titers to LBPA, but not prothrombin developed only in EPCR.sup.WT mice (FIG. 10E). In addition, only IgG from immunized EPCR.sup.WT mice induced TF activity and proinflammatory signaling in monocytes (FIG. 10F). Thus, EPCR is required for the development of autoimmunity in experimental APS.
Example 6: EPCR-LBPA Signaling Drives aPL Expansion and Autoimmune Pathology In Vivo
[0106] Specific inhibition of EPCR-LBPA completely prevented the development of aPLs (FIG. 11A) as well as double-stranded DNA autoantibodies, which were detectable already in 6-week-old MRL-Faslpr mice but not 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 from progressive kidney pathology as evidenced by reduced CD3+ and F4/80+ immune cell infiltration in the kidneys (FIG. 11C) and reduced renal pathology scores reflecting glomerular and interstitial damage (FIG. 11D).
[0107] In an independent 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 aged-matched MRL/MpJ control mice (FIG. 12A) and attenuated kidney infiltration of CD45+/F4/80+ immune cells measured by flow cytometry (FIG. 12B). These infiltrating myeloid cells expressed IFN-γ (FIG. 12C). Albuminuria only developed in mice treated with non-inhibitory αEPCR 1650, but not with inhibitory αEPCR-LBPA 1682 or in MRL/MpJ control mice (FIG. 12D). Thus, EPCR-LBPA signaling is crucial for both, the development of lipid-reactive antibodies as well as, more generally, drives kidney autoimmune pathology in this endosomal TLR7-dependent animal model.
REFERENCES
[0108] The references as cited herein are: [0109] 1. B. Giannakopoulos, S. A. Krilis, The pathogenesis of the antiphospholipid syndrome. N Engl J Med 368, 1033-1044 (2013). [0110] 2. K. Schreiber et al., Antiphospholipid syndrome. Nat Rev Dis Primers 4, 17103 (2018). [0111] 3. 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). [0112] 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). [0113] 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). [0114] 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). [0115] 7. D. Manukyan et al., Cofactor-independent human antiphospholipid antibodies induce venous thrombosis in mice. J. Thromb. Haemost 14, 1011-1020 (2016). [0116] 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 [0117] 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). [0118] 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). [0119] 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). [0120] 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). [0121] 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). [0122] 14. N. Müller-Calleja et al., Tissue factor pathway inhibitor primes monocytes for antiphospholipid antibody-induced thrombosis. Blood, (2019). [0123] 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). [0124] 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). [0125] 17. R. K. Sinha et al., Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization. Arterioscler. Thromb.
[0126] Vasc. Biol 36, 518-524 (2016). [0127] 18. F. 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). [0128] 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). [0129] 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). [0130] 21. G. Girardi et al., Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J. Clin. Invest 112, 1644-1654 (2003). [0131] 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). [0132] 23. A. S. Rothmeier et al., Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling. Blood 131, 674-685 (2018). [0133] 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). [0134] 25. I. Ott et al., Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor. Arterioscler. Thromb.
[0135] Vasc. Biol 20, 874-882 (2000). [0136] 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). [0137] 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). [0138] 28. T. Kobayashi et al., A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193-197 (1998). [0139] 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). [0140] 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). [0141] 31. P. Munzer et al., Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation. Arterioscler. Thromb. Vasc. Biol 34, 61-71 (2014). [0142] 32. H. Grassme et al., CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem 276, 20589-20596 (2001). [0143] 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). [0144] 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). [0145] 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). [0146] 36. B. Isermann et al., The thrombomodulin-protein C system is essential for the maintenance of pregnancy. Nat. Med 9, 331-337 (2003). [0147] 37. H. P. Liang et al., Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice. Blood 126, 2415-2423 (2015). [0148] 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). [0149] 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). [0150] 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). [0151] 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). [0152] 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). [0153] 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, [0154] 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). [0155] 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).
