Patent Application
20230043394
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.
Antiphospholipid syndrome (APS) is an acquired autoimmune disease in which a deficient control of the immune system leads to an increased tendency of the blood to coagulate. The resulting blood coagulation (thromboses) can subsequently lead to reduced blood flow (ischemia) to the affected tissue and trigger complications such as strokes, heart attacks or abortions. Although lipid-reactive antibodies also transiently appear in infectious diseases, clonal evolution of persistent antiphospholipid antibodies (aPL) in autoimmune diseases causes severe thrombo-embolic events, pregnancy morbidity, and fetal loss in the antiphospholipid syndrome (APS) (1).
Reactivity with cardiolipin is used to identify aPL, but aPL recognize a variety of anionic phospholipids and blood proteins, including β2-glycoprotein I (β2GPI). These complex reactivities have hampered the definition of a precise mechanism that causes the spectrum of APS-related pathologies (1, 2) and the development of autoimmune disease (3, 4). Clonal expansion of monoclonal aPL leads to protein cross-reactivity (5), but lipid recognition is sufficient to cause pregnancy complications (6) and thrombosis in mice (7), both of which involve a crosstalk of the innate immune defense complement and coagulation pathways (6, 8).
By binding to EPCR expressed by myeloid cells, aPL target a crucial toggle switch that controls coagulation and innate immune signalling. PAR2 activation by the TF-FVIIa-FXa-EPCR complex supports TLR4-mediated induction of interferon-regulated genes (16), but competition for EPCR ligand occupancy by the anticoagulant activated Protein C-FV-Protein S complex attenuates TF-dependent PAR2 signalling (37). Deregulated interferon signalling drives autoimmunity and by targeting EPCR aPL directly induce interferon signaling responses in myeloid cells, while mice with a disabled EPCR signalling pathway are protected from autoimmune aPL development.
Genetic or pharmacological inhibition of the antigenic target EPCR-LBPA attenuates aPL-induced pathologies in mice. Innate immune cell-expressed EPCR engagement by aPL induces interferon-regulated anti-microbial responses and drives interferon-dependent B cell expansion and the development of autoimmunity. Thus, aPL recognize a single lipid-protein receptor complex required for the pathogenesis and complications of this autoimmune disease.
US 2007-0141625A1 relates to a method for detecting autoantibodies against endothelial protein C/activated protein C receptor (EPCR) in a sample by its detection and in vitro quantification.
Sorice et al. (in: 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) disclose that anti-LBPA antibodies and IgG from APS patients affect the distribution of intracellular β2GPI in endothelial cell culture as well as the coagulation system. Further, they suggest that LBPA is a target for aPl and is involved in the immunopathogenesis of APS.
Alessandri et al. (in: Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. Clin Exp Immunol. 2005 Apr; 140(1):173-80. doi: 10.1111/j.1365-2249.2005.02727.x. PMID: 15762889) describe LBPA antibodies as biomarkers in antiphospholipid syndrome patients.
Olivieri et al. (in: 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) investigate anti-LBPA for its clinical value and reveals that anti-LBPA antibodies cannot be used to diagnose APS.
The prior art discloses that endothelial protein C receptor (EPCR) and lysobisphosphatidic acid (LBPA) or antibodies directed against endothelial protein C receptor (EPCR) or lysobisphosphatidic acid (LBPA) can be used as biomarkers to diagnose APS in patients. However, it is disclosed that antibodies against lysobisphosphatidic acid (LBPA) have no advantage as biomarkers compared to the analysis of other antibodies, e.g. against cardiolipin.
Thus, it is therefore an object of the present invention to provide a reliable and robust method for the detection of autoimmune disease, such as, for example, antiphospholipid syndrome (APS), in particular primary or secondary APS, based on binding of antiphospholipid antibodies (aPL).
A further object of the present invention is to provide a method for identification of potential inhibitors that prevent the aPL-pathogenic signalling.
Another object is the provision of a method for producing a pharmaceutical composition, wherein inter alia such an inhibitor is comprised.
Yet another object of the present invention is then to provide a method for treatment and/or prevention of an autoimmune disease, for example antiphospholipid syndrome (APS), in particular primary or secondary APS, in a subject by administering the pharmaceutical composition containing said inhibitor to said subject.
Other aspects and objects will become apparent to the person of skill upon studying the following description of the invention.
Surprisingly, the inventors in the context of the present invention identified endosomal lysobisphosphatidic acid (LBPA) and the presentation thereof by the CD1d-like endothelial protein C receptor (EPCR) as a so far unknown disease-causing cell surface antigen recognized by aPL. By intersecting with the innate immune and coagulation signalling function of EPCR, aPL engage with EPCR for endosomal trafficking and the initiation of prothrombotic and proinflammatory signalling.
The inventors were further able to show that the interaction of endothelial protein C receptor (EPCR) and LPBA is critical for the course of the antiphospholipid syndrome. This surprising discovery enables the use of the EPCR-LBPA complex as a novel target for diagnostic screening procedures and in screening procedures for the production of drugs that can be used to treat and prevent the antiphospholipid syndrome.
In a first aspect, the invention solves the above object by providing 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 said LBPA-binding fragment thereof, wherein said binding of aPL to said lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or an LBPA-binding fragment thereof detects an autoimmune disease in said subject.
In a second aspect, the invention relates to a method for identifying an inhibitor of endothelial protein C receptor (EPCR) function/activity in an autoimmune disease, preferably without interfering with its function in coagulation, comprising providing a biological sample comprising an EPCR protein or an lysobisphosphatidic acid (LBPA)-binding fragment thereof, contacting a potential inhibitor with said sample, and testing binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof in the presence or absence of said potential inhibitor, and identifying said potential inhibitor based on said LBPA-binding as tested.
In a third aspect, the invention relates to a method for identifying an inhibitor of endothelial protein C receptor (EPCR) function in autoimmune disease which preferably does not interfere with EPCR regulatory function in coagulation, comprising providing a biological sample comprising an EPCR protein or an lysobisphosphatidic acid (LBPA)-binding part thereof, binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA-complex, contacting a potential inhibitor with said sample, and testing binding of an antiphospholipid antibody (aPL) or cellular effects/functions in the presence or absence of said potential inhibitor, and identifying said potential inhibitor based on interference with said aPL-binding or cellular functions as tested.
In a fourth aspect, the invention relates to a method for producing a pharmaceutical composition, comprising the steps of identifying a potential inhibitor or inhibitor as described herein, and suitably formulating said potential inhibitor or inhibitor into a pharmaceutical composition.
In a fifth aspect, the 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.
In a sixth aspect, the invention relates to a method of treating and/or preventing an autoimmune disease, such as, for example, antiphospholipid syndrome, in particular primary or secondary APS, in a subject, said method 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.
In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
As mentioned above, in the first aspect thereof, the present invention relates to 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 an LBPA-binding fragment thereof detects the presence of an autoimmune disease in said subject.
An “LBPA-binding fragment” as used herein shall mean a part or fragment of the endothelial protein C receptor (EPCR) that is sufficient so that said LBPA-binding fragment is still capable of, preferably, binding lysobisphosphatidic acid (LBPA), i.e. the receptor affinity of the endothelial protein C receptor (EPCR) is retained by said LBPA-binding fragment. Included are also structural mimetics of such a binding domain. Preferably, all or part of the LBPA-binding fragment is produced recombinantly in expression suitable systems or by chemical synthesis.
While APS is the only manifestation of autoimmunity in many patients (primary APS), it also develops in in the context of other autoimmune diseases, in particular systemic lupus erythematosus (SLE) (secondary APS). Thus, preferred is an embodiment of the present invention, wherein the autoimmune disease is antiphospholipid syndrome, in particular primary or secondary APS. Further autoimmune diseases are selected from primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, without being limited to these.
As used herein, the term “antiphospholipid antibodies (aPL)” are autoantibodies which generally bind to negatively charged phospholipids, including cardiolipin (CL) as the antigen. Included are also antigen-binding fragments of these antibodies (see also below for further description).
As used herein, the term “binding” of aPL to said LBPA bound to EPCR or an LBPA-binding fragment thereof, or binding of LBPA to EPCR or an LBPA-binding fragment thereof, or further intermolecular bonds 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 in which they do not share electron pairs. Non-covalent interactions are classified in hydrogen bonds, Van-der-Waals interactions, hydrophobic interactions and electrostatic interactions. The presence of binding between the respective above-mentioned binding partners shall be investigated using “binding assays”, based on the specific binding respectively interactions between said binding partners. There are a number of suitable binding assays, such as an enzyme-linked immunosorbent assay (ELISA), that are known to the skilled person.
Further preferred is an embodiment of the present invention, wherein said lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or an LBPA-binding fragment thereof is immobilized, preferably directly or indirectly to a solid carrier material.
As used herein the term “directly” immobilized means the immobilization of an isolated and soluble endothelial protein C receptor (EPCR) or an isolated and soluble LBPA-binding fragment, wherein lysobisphosphatidic acid (LBPA) is bound thereto, and wherein said endothelial protein C receptor (EPCR) or said LBPA-binding fragment directly are immobilized covalent on the solid carrier material for example via photochemical methods. So-called photolinkers can be used, which are bound to the endothelial protein C receptor (EPCR) or said LBPA-binding fragment in order to fix the biomolecules covalently, parallel and directed on the solid carrier material. The photoreaction is triggered by UV irradiation, whereby the wavelength range above 300 nm must be used in order to avoid photolytic decomposition of the biomolecules. The photolinkers react with the substrate in a photoinduced radical reaction and the endothelial protein C receptor (EPCR) or said LBPA-binding fragment is directly immobilized on said solid carrier material.
The term “indirectly” immobilized means the fixation of cells expressing the endothelial protein C receptor (EPCR) or the LBPA-binding fragment, or part of cells presenting the endothelial protein C receptor (EPCR) or the LBPA-binding fragment on their surface on the solid carrier material, wherein lysobisphosphatidic acid (LBPA) is either already bound to the endothelial protein C receptor (EPCR) or the LBPA-binding fragment, or is added to the cell culture supernatant, so that the lysobisphosphatidic acid (LBPA) bound to endothelial protein C receptor (EPCR) or the LBPA-binding fragment is provided via fixed cells or parts thereof on their surface, wherein the cells or parts thereof are fixed on the solid carrier material. The term “cells” used in the context of the present invention means eukaryotic cells capable of expressing the endothelial protein C receptor (EPCR) or the LBPA-binding fragment. Therefore, the PROCR-Gene encoding the endothelial protein C receptor (EPCR) or a nucleic acid encoding the LBPA-binding fragment can either already be present in the cells or the cell can be transfected with the nucleic acids or a vector comprising the nucleic acids. The term “eukaryotic” includes yeast, higher plant, insect and mammalian cells. Once the nucleic acid or vector has been transfected into the corresponding cell, the cell is kept under conditions suitable for high-grade expression of the nucleic acids or the vector.
The term “solid carrier material” shall refer to any solid support material which is chemically inert and allows the direct or indirect immobilization of the endothelial protein C receptor (EPCR) or the LBPA-binding fragment to the solid support material. A large immobilization area can be achieved by using very porous materials. Furthermore, the carrier must allow substances used in the context of the present invention to flow in and out. A number of suitable carriers are known. The solid carrier material can be, for example, selected from glass, agarose, polymers, or metals, but without being limited to it.
In the second aspect, the invention relates to a method for identifying an inhibitor of endothelial protein C receptor (EPCR) function in an autoimmune disease while preferably not interfering with EPCR regulatory function in coagulation, comprising providing a biological sample comprising an EPCR protein or an lysobisphosphatidic acid (LBPA)-binding fragment thereof, contacting a potential inhibitor with said sample, and testing binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof in the presence or absence of said potential inhibitor, and identifying said potential inhibitor based on said LBPA-binding as tested. This assay therefore seeks to identify inhibitors of the binding between LBPA to the EPCR protein.
In the third aspect, the invention relates to 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 providing a biological sample comprising an EPCR protein or an lysobisphosphatidic acid (LBPA)-binding fragment thereof, binding of LBPA to said EPCR protein or said LBPA-binding fragment thereof to form an EPCR-LBPA-complex, contacting a potential inhibitor with said sample, and testing binding of an antiphospholipid antibody (aPL) or cellular functions in the presence or absence of said potential inhibitor, and identifying said potential inhibitor based on interfering with said aPL-binding or cellular effects/functions as tested. This assay therefore seeks to identify inhibitors of the binding between the LBPA/EPCR protein complex, and the aPL, and “general” inhibitors interfering with the signalling pathway involving said complex and aPL
In addition to “binding” as described above, potential inhibitors can also be identified via “cellular functions” within intact cells present in the biological sample. “Cellular functions”, as used in the context of the present invention, are based on alterations in protein expression of interferon induced genes in said cells present in the biological sample in the presence or absence of the potential inhibitor. For example, both inhibitory and non-inhibitory binding partners are able to bind to the LBPA-EPCR complex, EPCR or aPL. While binding of an inhibitory binding partner, i.e. a potential inhibitor, prevents the aPL-induced interferon response, the binding of non-inhibitory binding partners does not alter the aPL-induced interferon response. The interferon response then leads to expansion of aPL producing B-cells and expression of interferon-induced genes. Interferon-induced genes comprise, but are not limited to, IRF8, GBP2, GBP6.
Preferred is an embodiment of the method according to the present invention, wherein at least one of EPCR, fragment, LBPA, said potential inhibitor and/or aPL is suitably labelled and/or immobilized.
The term “suitably labelled” as used herein means that at least one of EPCR, fragment, LBPA, said potential inhibitor and/or aPL may contain additional markers, such as non-protein molecules such as nucleic acids, sugars, or markers for radioactive or fluorescent labelling. The label is either directly or indirectly involved in generating a detectable signal.
In another preferred embodiment of the present invention, the method further comprises the step of testing said potential inhibitor as identified for being an inhibitor of endothelial protein C receptor (EPCR) function in an autoimmune disease without interference of EPCR function as a regulator of coagulation. The inventors showed that binding of aPL to the EPCR-LBPA complex leads to the internalization of the complex and pathogenic aPL signalling. The important function of EPCR as a regulator of coagulation is maintained, as EPCR binds to its agonist protein C in the absence of LBPA, wherein binding of protein C to EPCR is not prevented by the inhibitors as identified. This testing can also involve the other components of the system, LBPA, and/or aPL.
Using the term “suitably testing” in the context of the present invention, a distinction is made between suitable testing of the binding or of suitable testing of the cellular functions. A suitable testing of the binding means the detection of a generated detectable signal depending on the used label with a suitable detection system to determine whether a potential inhibitor could prevent the binding of LBPA to EPCR or the LBPA-binding fragment, or whether a potential inhibitor could prevent the binding of aPL to the LBPA-EPCR complex. For example, FRET probes can be used for suitable testing, where one binding partner is labelled with a donor fluorochrome and another binding partner is labelled with an acceptor fluorochrome. The fluorescence signal emitted can be used for very specific detection of whether the potential inhibitor to be identified prevented binding of the binding partners involved. Many other detection systems are known in the prior art. Suitable testing of the cellular functions means the detection of aPL induced interferon response or the detection of expressed aPL due to expanded B cells. The detection can be performed at the posttranscriptional or posttranslational level either by quantification of mRNA or proteins. The skilled person is aware of methods for mRNA and protein analysis.
Further preferred is an embodiment of the method according to the present invention, 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.
The term “small molecule” as used herein describes a class of substances with a low molecular mass, that does not exceed about 900 Dalton. Due to their small size, small molecules are partly able to penetrate into cells. Small molecules can be chemically synthesized. The term covers an extremely heterogeneous group of substances. Small molecules have a multitude of biological functions, such as signal molecules. They can be of natural (e.g. secondary metabolites) or artificial (e.g. antivirals) origin. Some small molecules are able to cross the blood-brain barrier.
The term “protein” is used to denote a polymer composed of amino acid monomers joined by peptide bonds. It refers to a molecular chain of amino acids, and does not refer to a specific length of the product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation. Amino acid chains with a length of less than approx. 100 amino acids are called “peptides”. The terms “peptides”, and “proteins” as used herein are included within the definition of “polypeptides”. A “peptide bond” is a covalent bond between two amino acids in which the α-amino group of one amino acid is bonded to the α-carboxyl group of the other amino acid. All amino acid or polypeptide sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).
“Antibody” and “antibodies” refer to antigen-binding proteins that arise in the context of the immune system. The term “antibody” as referred to herein includes whole, full length antibodies and any fragment or derivative thereof in which the “antigen-binding portion” or “antigen-binding region” or single chains thereof are retained, such as a binding domain of an antibody specific for lysobisphosphatidic acid (LBPA), endothelial protein C receptor (EPCR), LBPA-binding fragment, LBPA-EPCR-complex, or antiphospholipid antibodies (aPL). A naturally occurring “antibody” (immunoglobulin) is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised 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's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to 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 referred to as the variable (Fv) region, and the Fc (fragment, crystallizable) region. The variable regions (Fv) of the heavy and light chains contain a binding domain that interacts with an antigen. The constant (Fc) regions of the antibodies may mediate the 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 mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns. One suitable Fc polypeptide is derived from the human IgG1 antibody.
Fragments, derivatives, or analogs of antigen-binding proteins such as antibodies can be readily prepared using techniques well-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 as compared to a corresponding full-length antigen-binding protein. Examples of fragments of antigen-binding proteins encompassed within the term “antigen-binding fragments” include a Fab fragment; a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab′)2 fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment which consists of a VH domain; an isolated complementarity determining region (CDR); and a single chain variable fragment (scFv). An antigen-binding protein or fragment or derivative thereof or fusion protein thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific antibody” or “bifunctional antibody” has two different binding sites. Bispecific antibodies are preferred molecules of the invention and may be selected from any bispecific format known to the skilled artisan such as bites or diabodies. A “derivative” of an antigen-binding protein is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety (such as, for example polyethylene glycol or albumin, e.g., human serum albumin), phosphorylation, and/or glycosylation.
An “scFv” is a monovalent molecule that can be engineered by joining, using recombinant methods, the two domains of the Fv fragment, VL and VH, by a synthetic linker that enables them to be made as a single protein chain. Such single chain antigen-binding peptides are also intended to be encompassed within the term “antigen-binding portion.” 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 are intact antibodies.
The term “antigen-binding fragment” or “antigen-binding region” of an antigen-binding protein such as an antibody, or grammatically similar expressions, as used herein, refers to that region or portion that confers antigen specificity; fragments of antigen-binding proteins, therefore, include one or more fragments of an 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.
The term “enzyme” as used herein refers to a protein with catalytic activity.
As used herein the term “aptamer” means short single-stranded DNA or RNA oligonucleotides (25-70 bases) that can bind to a specific molecule. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sides.
Preferred is an embodiment of the method according to the present invention, wherein said subject is a mammal, preferably a human.
Further preferred is an embodiment of the method according to the present invention, wherein said biological sample is selected from a body fluid, including blood, serum, and saliva, and a tissue, organ or cell type blood sample, a sample of blood lymphocytes and a fraction thereof.
In the fourth aspect, the invention relates to a method for producing a pharmaceutical composition, comprising the steps of identifying a potential inhibitor or inhibitor as described herein, and suitably formulating said potential inhibitor or inhibitor into a pharmaceutical composition.
As used herein the term “pharmaceutical composition” refers to a “suitable formulation” which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled person, the route and/or mode of administration will vary depending upon the desired results. To administer a binding compound according to the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
An “appropriate carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. Appropriate carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Administration may be, for example, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). The prevention of the presence of microorganisms can be ensured both by sterilization procedures, Supra, and by 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 sugar, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable dosage form can be achieved by using absorption retardants such as aluminium monostearate and gelatin.
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, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skilled in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride are included in the composition.
The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 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, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
In the fifth aspect, the 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 while preferably avoiding interference with vascular protective functions of EPCR.
As used herein, the terms “preventing” or “prevention” comprise the administration of said compound(s) to said subject, preferably in a preventively effective amount to refer to reduce, no matter how slight, of a subject's predisposition or risk for developing an autoimmune disease, such as an antiphospholipid syndrome, in particular primary or secondary APS, primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis. For prevention, the subject is preferably a subject who is at risk or susceptible to the development of an autoimmune disease, such as an antiphospholipid syndrome, in particular primary or secondary APS, primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
The terms “treating” or “treatment”, as used herein, comprise the administration of said compound(s) to said subject, preferably in a therapeutically effective amount to alleviate the disease or progression of an autoimmune disease, such as an antiphospholipid syndrome in particular primary or secondary APS, primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
Preferred is an embodiment of the present invention, wherein the inhibitor or pharmaceutical composition for use, as described herein, is selected from a small molecule, a peptide, an antibody or antigen-binding fragment thereof, an enzyme, and an aptamer.
Further preferred is an embodiment, wherein said autoimmune disease is an antiphospholipid syndrome (APS), in particular primary or secondary APS, primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.
In the sixth aspect, the invention relates to a method of treating and/or preventing an autoimmune disease, such as, for example, antiphospholipid syndrome, in particular primary or secondary APS, primary Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis, in a subject, said method 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.
The terms “administering” or “administration” used herein cover enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastern injection and infusion.
An “effective amount” as used herein, is an amount of the compound(s) or the pharmaceutical composition(s) as described herein that normalize the inflammatory state in the subject. The amount alleviates symptoms as found for the disease and/or condition, without being toxic to the subject. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range). However, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and/or claimed herein.
In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a 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 larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a 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 larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.
It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.
All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.
The figures show:
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.
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 (
EPCR blockade similarly inhibited procoagulant and proinflammatory aPL responses in human monocytes (
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 EPCRC/S monocytes (
Imaging demonstrated that aPL HL5B did not bind to EPCR-deficient (EPCRlow) monocytes (18) or cells blocked by the inhibitory αEPCR 1560, whereas the non-inhibitory αEPCR 1562 prevented neither binding nor aPL internalization (
Certain aPL interfere with anticoagulation (24), but this feature was not common to all lipid-reactive prototypic aPL (
αEPCR 1682 surprisingly did not stain EPCR that was expressed at normal levels on monocytes from EPCRC/S mice (
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 (
Importantly, simply adding LBPA to the culture medium of EPCRC/S, but not EPCR-deficient cells restored cell surface αLBPA 6C4 and αEPCR 1682 staining (
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 (
Competition experiments confirmed the high affinity of aPL HL5B for LBPA-loaded sEPCR (
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 (
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 (
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 (
Importantly, thrombosis induction by aPL HL5B was markedly reduced in EPCRC/S as compared to strain-matched WT controls (
The importance of this pathway in human trophoblast cells by knock-down of ALIX (
Human ALIX−/− trophoblast cells and murine EPCRC/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−/− trophoblast cells (
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 EPCRC/S mice or EPCRlow mice relative to WT controls (
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 EPCRC/S monocytes and, as shown for GBP2, LBPA addition restored interferon responses (
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 (
Supporting this conclusion, anti-cardiolipin antibody production was absent when mouse pDC, but not B cells, were isolated from EPCRC/S mice (
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−/− mice displayed a slightly enhanced response (
APS is also triggered by immunization with human β2GPI (45) which induced a similar high titer IgG antibody response to human β2GPI in EPCRWT and EPCRC/S mice (
Specific inhibition of EPCR-LBPA completely prevented the development of aPLs (
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 (
The references as cited herein are:
Vasc. Biol 36, 518-524 (2016).
Vasc. Biol 20, 874-882 (2000).
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/085278 | 12/9/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62955060 | Dec 2019 | US |
