EP2344209A2

EP2344209A2 - Cfhr1 as inhibitor of c5 convertase for the treatment of autoimmune diseases

Info

Publication number: EP2344209A2
Application number: EP09736819A
Authority: EP
Inventors: Peter Zipfel; Christina Skerka; Reinhard Wallich
Original assignee: Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie Ev -Hans-Knoll-Institut- (hki); Leibniz Institut fuer Naturstoff Forschung und Infektionsbiol eVi
Current assignee: Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie Ev -Hans-Knoll-Institut- (hki); Leibniz Institut fuer Naturstoff Forschung und Infektionsbiol eVi
Priority date: 2008-09-26
Filing date: 2009-09-28
Publication date: 2011-07-20
Also published as: WO2010034514A2; DE102008049136A1; WO2010034514A9; US20110236455A1; WO2010034514A3; DE102008049136B4

Abstract

The invention relates to the identification of new regulators of the innate immune system, in particular the complement system. More particularly, the invention relates to specific C5 convertase inhibitors. Said novel inhibitors are particularly useful for treating inflammatory diseases in which the complement system is involved. In a first embodiment, the invention is directed to the use of CFHR proteins and functional fragments or functional derivatives thereof for preventing inflammatory reactions. In another embodiment, the invention is directed to the use of said CFHR proteins for preventing complement activation during transplantations and dialyses and for coating devices that enter in contact with blood or body fluids, especially implants. According to the invention, a pharmaceutical composition comprising functional CFHR protein in combination with functional factor H is also prepared. In yet another embodiment, the invention is directed to the preparation of monoclonal antibodies which specifically detect CFHR proteins, and the use thereof in methods for determining CFHR in body fluids. Said methods are particularly suitable for diagnosing inflammatory diseases.

Description

New regulators of the innate immune system

The present invention relates to the identification of novel regulators of the innate immune system, in particular the complement system. More particularly, the present invention relates to specific inhibitors of C5 convertase. These new inhibitors are particularly useful for the treatment of inflammatory diseases involving the complement system. In a first aspect, the present invention is directed to the use of CFHR proteins and functional fragments or functional derivatives thereof for the prophylaxis of inflammatory responses. In another aspect, the present invention is directed to the use of CFHR proteins to inactivate complement activation during transplantation, dialysis, and for coating devices in contact with blood or body fluids, particularly implants. The invention further provides a pharmaceutical composition comprising functional CFHR protein in combination with functional factor H. In another aspect, the present invention is directed to the provision of monoclonal antibodies that specifically detect CFHR proteins and their use in methods for the determination of CFHR in body fluids. These methods are particularly suitable for the diagnosis of inflammatory diseases.

State of the art

The complement system is an important element for innate and acquired immunity and is essential for eliciting a protective immune response to a foreign invader. The alternative pathway of the complement system is activated spontaneously and involves the formation of the C3 convertase (C3bBb), which cleaves the central component of the complement system, C3. This cleavage generates the anaphylactic peptide C3a and the active protein or activation product C3b, which can attach to a surface. C3b, attached to foreign or altered surfaces, binds factor B and forms the C3 convertase (C3bBb). This enhances further complement activation, which eventually leads to opsonization and phagocytosis of invading objects such as microbes. The binding of a second C3b molecule to the C3 convertase leads to the formation of the C5- Convertase (C3bBbC3b) of the alternative route. The C5 convertase cleaves C5 and generates the potent chemoattractant C5a and the peptide C5b. This peptide C5b initiates the formation of the terminal membrane-attacking complex (MAC). C5b immediately binds to C6 and C7 in an enzyme-independent manner due to conformational changes. This formed C5b67 complex dissolves from the convertase and attaches to lipid bilayers. Upon binding of C8 and C9, the complete terminal membrane-attacking complex is formed which results in lysis of the pathogen and cells.

Once activated, this defense system is tightly regulated at the surface of host cells by both membrane-anchored and soluble regulators, which attach in both the liquid phase and the surface. To ensure no negative effects against own tissue and own cells, this strict regulation is necessary. Single mutations in genes encoding the appropriate regulators of the host cells and resulting in defective protein functions are predisposing to various immune deficiency and autoimmune diseases as well as renal and retinal diseases, e.g. hemolytic uremic syndrome (HUS), membran proliferative glomerulonephritis type II (MPGN II) or age-related macular degeneration (AMD).

It is known that these different diseases are caused by defective local complement regulation and are associated with gene variations and mutations in complement components and regulators such as CFH (complement factor H). Thus, deletion of a 84 kb genomic fragment on human chromosome 1 resulting in loss of complement factor H related genes 1 and 3 (complement factor H related genes 1 and 3 (CFHR1, CFHR3)) has been shown to occur with both HUS and AMP (Zipfel PF et al., PLoS .3: E41 (2007), Hughes AE et al., Nat. Genet., 38, 1173-1177 (2006)). However, these chromosomal deletions showed opposite effects, so in HUS the deletion led to an increased risk for the disease, whereas in AMD a protective effect is described. The absence of these two plasma proteins CFHR1 and CFH3 further correlates with the presence of autoantibodies to CFH (Jozsi, M. et al., Blood, 111, 1512-1514 (2008).) These identified autoantibodies bind to the C-terminal end of CFH This area is a focal point of HUS mutations and leads to a reduced attachment of CFH to the surface. A corresponding autoantibody binding to CFH thus leads to the inhibition of

Adhesion of CFH to the surface resulting in damage to

Endothelial cells, as well as platelets.

The family of CFHR proteins currently comprises five members in humans, CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5. CFHR genes and proteins also occur in other species. In the mouse and rat these are e.g. designated CFHR-A, CFHR-B, CFHR-C, etc.

These members of the CFHR protein family are all characterized by a very similar structure, similar in structure modules and high sequence homology between the individual modules to each other. However, every CFHR will

Protein encoded by a specific unique gene. The function of the individual CFHR proteins is unknown. It is merely shown that the CFHR

Proteins have no cofactor activity and decay activity.

The CFHR proteins also have a high sequence homology to the complement factor H (CFH). In particular, in the C-terminal region, e.g. CFHR1 with its three SCR (short complement regulator) domains has a high homology varying from almost 100% to a low, approximately 65% identity with the corresponding SCR domains at the C-terminal end of the complement factor H on. The CFHR1 plasma protein is composed of 5 of these SCR domains and has been identified in two glycosylated forms in human plasma.

CFHR1-beta has two and CFHR1-alpha has an attached sugar side chain.

The function of CFHR-1 and the related molecules CFHR2, CFHR3,

CFHR4 and CFHR5 are unknown. So far it has been speculated that the molecules could have the following functions: binding to C3b and to heparin and a modulating influence on the regulatory function of factor H.

As stated above, the C5 convertase is an essential target for the

Inhibition of complement activation, since both the resulting from the C5

Anaphylatoxin C5a is necessary for triggering a local inflammatory response in an infection, as well as the resulting peptide C5b, which together with C6 attack the initial complex for the formation of the terminal membrane

Complex forms. Inhibitors that specifically inhibit this enzyme are not previously known and therefore are particularly suited to inhibit the corresponding subsequent alternative complement activation.

An object of the present invention is to provide specific inhibitors of C5 convertase so as to provide the alternative route, i. the

Inhibiting complement activation, including the formation of terminal membrane-attacking complexes, as well as inhibiting the formation of effective anaphylactic, and possibly antimicrobial, peptides

C5a. This specific inhibition of C5 convertase should preferably not affect the classical pathway or lectin pathway of complement activation.

In addition, inhibition of terminal complement activation in the form of the formation and assembly of the terminal complement complex (MAC, membrane attack complex or TCC) and its incorporation into the lipid bilayer membrane is of great interest. At present, two terminal complement pathway inhibitors are known, clusterin and vitronectin. However, the specificity of these regulators is not very high, so it makes sense to use more specific inhibitors.

Another object of the present invention is to provide

Detecting agents, in particular antibodies that specifically allow the detection of CFHR molecules. Finally, another objective of the present

Invention is the provision of agents for the treatment of inflammation as well

Method for this.

Summary of the Present Invention The present invention provides the use of CFHR proteins, in particular CFHR1 proteins, or functional fragments or functional derivatives thereof for the treatment or prophylaxis of autoimmune diseases or inflammatory reactions.

It has been found herein that CFHR proteins are specific inhibitors of C5 convertase. By specifically inhibiting the C5 convertase, both the formation of the anaphylatoxin C5a and the formation of the terminal membrane-attacking complex can be inhibited. The CFHR proteins are specific C5 convertase inhibitors that are not C3 convertase, in contrast to known C3 / C5 convertase inhibitors such as CFH, inhibit. That is, for the first time, a C5 convertase-specific inhibitor will be described. These specific inhibitors allow modulation of the alternative pathway of complement activation without significantly affecting, eg blocking, the classical pathway. In one aspect, the present invention is directed to the use of these functional CFHR proteins and their functional fragments and derivatives to inactivate complement activation, particularly in transplantation or dialysis. In another aspect, these functional CFHR proteins can be used to coat surfaces that may come in contact with blood and body fluid, such as implant surfaces.

In another aspect, appropriate coatings and devices are provided.

Furthermore, the present invention is directed to a pharmaceutical composition comprising functional CFHR protein in combination with functional factor H.

Finally, a monoclonal antibody is provided which specifically recognizes CFHR proteins. In particular, this monoclonal antibody allows the specific recognition of CFHR protein, such as CFHR1 protein compared to complement factor H. Finally, methods for the determination of CFHR in

Body fluids, especially in blood and blood plasma, comprising the use of the antibody of the invention, in particular for the diagnosis of hemolytic uremic syndrome, age-related macular degeneration or membranoproliferative glomerulonephritis but also atherosclerosis and other autoimmune diseases such as systemic lupus erythematosus.

Brief description of the illustrations

Figure 1: Figure 1 shows the ability of CFHR1 to bind C3b, C3d, heparin and human cells. Figure 1a shows the construction of CFHR1 compared to complement factor H. The first two SCHRs of CFHR1 show 42 and 34% sequence identity, respectively, with SCHs 6 and 7 of CFH. The three C-terminal SCRs of CFHR1 show 100, 100 and 98% sequence identity, respectively, to SCHs 18 to 20 of CFH. Figure 1b shows that equimolar concentrations of CFHR1 and CFH bind to immobilized C3b. The data shows the average values plus minus Standard deviations from one of three independent experiments. A490: absorbance at 490 nm. Co: control. FIG. 1c shows the binding of CFHR1 or CFH to immobilized heparin. The control represents the background binding of the antibodies in the presence of the buffer. Figure 1d shows the CFHR1 binding to HUVEC cells, with the CFHR1 protein being obtained from plasma. The cells were incubated in human plasma and bound CFHR1 was detected by flow cytometry using the monoclonal antibody JHD10. The control cells were treated with secondary antibodies alone. Figure 1e shows the binding (10 μg / ml) of recombinant CFHR1 to HUVEC cells and to retinal pigment epithelial cells; the line means a size of 20 microns. Similarly, the CFHR1 binding was studied in rabbit erythrocytes pretreated with C3b.

Figure 2 shows the CFHR1 staining on tissue sections of the kidney and choroid. I shows the staining in the kidney, Il in the eye. The counterstain is with propidium iodide.

Figure 3 illustrates the specificity of the monoclonal antibody JHD10 described herein, which specifically recognizes CFHR1 in human serum and does not cross-react with CFH. Figure 3a shows the specificity of the monoclonal antibody. Lane 1 normal human serum, lane 2 CFH, lane 3 normal human serum, lane 4 CFH. While the CFH-specific antibody detects CFH, the monoclonal antibody JHD10 shows reactivity only to the CFHR1 molecules. Figure 3b shows silver staining of the purified recombinant CFHR1 (lane 1) and CFHR fragments CFHR1 / 1-2 (lane 2) and CFHR1 / 3-5 (lane 3). FIG. 3c shows recombinant CFHR1 and native CFHR1 obtained from plasma (lanes 1 and 3 or 2 and 4). The right side shows an immunoblot using the specific CFHR1 antibody, the left side showed silver staining.

In Figure 4 it is demonstrated that CFHR1 competes with factor H for binding to the binding partners. Figure 4a shows immunofluorescent images showing that CFH and CFHR1 colocalize (yellow signal resulting from green fluorescence for CFH and red fluorescence for CFHR1). Figure 4b shows the competition of CFHR1 with factor H for C3 binding. The data represent averages of three separate experiments plus minus standard deviations. A490: absorbance at 490 nm; * p <0.05 compared Binding of CFHR1: CFH (0: 1). Figure 4c shows the competitive binding of CFHR1 with factor H in heparin. Shown is the mobility of the alpha 'and beta chain as well as the degradation fragments.

FIG. 5 shows the results for the regulation of the alternative complement pathway by CFHR1. Figure 5a shows the hemolysis of sheep erythrocytes in the presence of CFHR1 and CFH depleted normal human serum (HPΔCFH-CFHR1). For comparison, the results with vitronectin, CFH and HSA. A dose-dependent reduction in lysis was shown by the addition of CFHR1. FIG. 5b shows the inhibition of the alternative complement pathway by CFHR1. Using an ELISA, each of the three complement pathways was induced separately. AP alternative route, CP classical route, LP lectin route; A440: absorption at 440 nm. For the alternative route, a dose-dependent effect could be shown for CFH R1 (squares). Figure 5c shows that CFHR1 does not affect the formation of C3a, unlike CFH. FIG. 5d shows the protective effect of CFHR1 and the inhibition of C5bC6 generation. Rabbit erythrocytes were incubated with human complement active CF7 depleted CFHR1 / CFHR3 deficient human plasma. CFHR1 was then added in appropriate concentration. Lysis was carried out using chicken erythrocytes to which sub-lytic amounts of human plasma as a source of C7 were added. The erythrocyte lysis was determined after 15 minutes of incubation.

Figure 6 shows that CFHR1 regulates and binds C5 convertase activity to C5 and C5b6, as well as inhibiting the binding of C5b6 to cell surfaces and thus the formation of the MAC and the enzymatic activity of the MAC. FIG. 6a shows the effect of CFHR1 on the lysis of sheep erythrocytes and the formation of C3a and C5a in the supernatant of the sample. The data represent averages of three separate experiments and their standard deviations. ^* : P <0.05, ^** : p <0.005 compared to the control. In Figure 6b, the effect of CFHR1 on the deposition of C3b and C5b on the surface of sheep erythrocytes is determined. Figure 6c shows immunofluorescent images showing that CFHR1 inhibits attachment of C5b to cell surfaces while CFH inhibits attachment of both C3b and C5b. FIG. 7 shows the binding of CFHR1 to C5 or C5b6 and the inhibition of the terminal complement pathway. FIG. 7a shows the binding of CFHR1 to immobilized C5 or C5b6. As a control, the binding of the antibody JHD10 to C5 or C5b6 alone was determined. FIG. 7b shows that the binding of C5 or C5b6 takes place via the N-terminal region of CFHR1. ^** p <0.001 versus the binding of C5 or C5b6 to C18-mediated CFHR1 immobilization. In Fig. 7c, chicken erythrocytes were incubated with C5b6 complexes (5 ng / ml), increasing concentrations of CFHR1 and non-lytic HP were added. There is a dose-dependent inhibition of lysis on addition of CFHR1, while CFH and HSA controls do not limit lysis. Figure 7d shows the inhibition of CFHR1 lysis of sheep erythrocytes compared to CFH and BSA. Similar values were found for the known inhibitor vitronectin (Vitro). The data represent mean values plus minus standard deviations of 2 separate experiments.

Detailed description of the invention

The present invention relates, in a first aspect, to the use of functional CFHR proteins for the treatment or prophylaxis of autoimmune diseases and inflammatory reactions.

In this context, the term "CFHR functional proteins" means both the CFHR protein itself and functional fragments of this protein, as well as functional derivatives of the CFHR protein which, in their function of inhibiting the C5 convertase, as shown herein, the overall The term CFHR protein hereby includes the human CFHR proteins CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, the individual proteins having the following accession numbers: CFHR1 NM 002113 gi 118442838, CFHR2 NM 005666 gi49574530, CFHR3 NM 021023 gi 118421081, CFHR4 NM 006684 gi 117320517, CFHR5 NM 030787 gi 164607154. In the following, the term "functional CFHR protein" is understood to mean both the protein and the functional derivatives and functional fragments, if stated differently. However, preference is given to the use of the mature CFHR protein. "Functional" here means that the CFHR molecules, in particular the CFHR proteins but also the fragments or derivatives of the CFHR proteins have an inhibitory effect on the C5 convertase and optionally inhibits the formation and activity of the MAC without the C3 convertase of the complement system In this case, the functional fragment preferably has at least 60%, such as 70%, in particular 80%, preferably 90%, activity with respect to the inhibitory effect on the C5 convertase in comparison to the total -CFHR protein, especially the CFHR1 protein.

Fragments include those derived from the CFHR protein polypeptides having at least one deletion, mutation or addition of an amino acid.

Derivatives of the CFHR protein are polypeptides obtained by post-translational modification of the CFHR protein or CFHR fragment, e.g. Glycosylation, acetylation, phosphorylation and the like. These derivatives further include those polypeptides in which one or more analogs of amino acids, including non-naturally occurring amino acids, polypeptides having substituted linkages such as PNA polypeptides, and other modifications occur naturally or non-naturally.

Unless otherwise stated, the term "complement activation" refers on the one hand to the formation of the terminal membrane attacking complex, hereinafter also referred to as MAC (membrane attacking complex) On the other hand, the term "complement activation" encompasses the generation of inflammation-promoting peptides, in particular anaphylatoxins, such as C5a. The term "bodily fluids" refers to fluids of the body including blood, blood plasma, lymph, cerebrospinal fluid, cerebrospinal fluid, synovial fluid, etc.

According to the invention, the CFHR protein is preferably the CFHR1 protein. It was shown that functional CFHR proteins show an inhibitory effect specific to the C5 convertase. The C5 convertase cleaves the complement C5 into the peptide components C5a and C5b. C5a acts as an inflammatory-mediating anaphylatoxin that locally induces or enhances inflammatory responses. A C5b molecule binds to a C6 molecule and this C5b, 6 complex then attaches to a molecule of C7. This reaction leads to a confirmation change in the molecules involved, with a hydro site on C7 becoming accessible. This hydrophobic domain of C7 pushes into the lipid bilayer. Hydrophobic sites are similarly exposed to later components C8 and C9 when they bind to the complex; this also allows them to penetrate into the lipid bilayer. The next step is the attachment of a C8 molecule to the membrane-associated C5b67 complex. C8 is a complex of two proteins: Cδbeta, which binds to C5b, and Cδalpha-gamma, which penetrates into the lipid bilayer. Finally, Cδalpha-gamma induces the polymerization of 10 to 16 C9 molecules into an annular structure called a terminal membrane-attacking complex. So the cell or the pathogen can be destroyed eventually.

In the present case it now appears that CFHR protein, in particular the CFHR1 protein, can inhibit the activity of the C5 convertase and furthermore the formation of C5a and C5b. Furthermore, it was found that the CFHD protein according to the invention does not inhibit the activity of C3 convertase. Finally, the CFHR protein inhibits the formation of the C5Bb6 (7) complex and thus the formation of the MAC. This feature allows the use of the functional CFHR protein for the treatment or prophylaxis of inflammatory reactions. The inflammatory reactions are preferably inflammatory reactions in the context of an autoimmune disease

These autoimmune diseases, which may include autoantibodies, are particularly those such as rheumatoid arthritis, lupus erythematosus, hemolytic uremic syndrome, atherosclerosis, renal diseases such as glomerulonephritis and others.

Furthermore, proteinuria-related diseases can be treated, such as proteinuria in kidney disease.

The inflammatory reactions to be treated are particularly preferably those in the context of sepsis, rheumatoid arthritis, Alzheimer's disease, atherosclerosis, lupus erythematosus, antiphospholipid syndrome, preclampsia, multiple sclerosis, myocarditis, asthma, recurrent pregnancy loss syndrome.

In a further aspect, the CFHR proteins are useful for inactivating complement activation, and in particular for inactivating the complement Complement activation in transplantation or dialysis. Since the deficiency of CFHR1 leads to kidney damage, and in a transplantation the activated complement system plays an essential role in graft acceptance, specific inhibitors that allow for specific inhibition of complement activation are therapeutically desirable.

Finally, further use of the CFHR protein involves the coating of devices and surfaces associated with bodily fluids, and particularly blood or blood plasma. By coating the devices, complement activation in the body fluid can be prevented by these devices.

Therefore, another aspect of the present invention is directed to a coating of a device in contact with body fluids, particularly blood or blood plasma, characterized in that the surface is coated with CFHR functional proteins. This coating is particularly suitable for implantable

Devices, wherein this implantable device is intended for use on the body of an individual.

Another aspect of the present invention is directed to such devices that come into contact with body fluids, such as blood or blood plasma in particular. This device, which is in particular an implantable device, is characterized in that functional CFHR protein has been applied to the surface.

CFHR1 as an example of CFHR proteins shows an inhibitory effect on the complement activation of the alternative pathway. It turned out that this effect is not due to inhibition of C3 convertase, although CFHR1 competes with complement factor H (CFH) for binding to C3b and can partially replace CFH. On the contrary, inhibition of C3 convertase is not observed. Experiments showed that CFHR1 is able to specifically regulate the activation of the alternative complement pathway. It could be a CFHR1 dose-dependent inhibitory effect on the

Complement-mediated lysis can be demonstrated by formation of the terminal complex and MAC formation. That is, the CFHR molecule of the present invention can inhibit the formation and activity of MAC. Finally, it was shown that CFHR1 can inhibit the alternative pathway but also the classical and the lectin pathway via the C5 convertase and the MAC.

The administration of CFHR1 resulted in downregulation of C5a peptides and C5b proteins, but the generation of C3a and C3b was unaffected. In contrast, the effect of CFH _{1 on} both the

C3 convertase as well as the C5 convertase inhibits and thus both the

Generation of C3a and C3b as well as C5a and C5b inhibits. That is, the

CFHR proteins show an activity profile different from CFH, acting only as an inhibitor of C5 convertase, while CFH acts on the cascade C3 convertase and C5 convertase.

In fact, CFHR1 can bind to C5 and the C5b6 complex. This inhibits the formation of the MAC and prevents lysis of cells or microbes. Thus, CFHR1 was shown to be the alternative pathway by inhibiting the

Regulates C5 convertase activity, MAC confluence, and preventing surface binding.

Another aspect of the present invention is directed to supplementing CFH therapy with CFHR proteins. The two proteins are important regulators of the complement cascade, leading to different procedures in the complement system.

Another aspect of the present invention is thus directed to a pharmaceutical composition comprising functional CFHR protein.

Preferably, the pharmaceutical composition further comprises the functional complement factor H, i. In a preferred embodiment, the pharmaceutical composition is a broadly functional CFHR protein in

Combination with functional complement factor H. The pharmaceutical

Composition also optionally contains conventional pharmaceutically acceptable diluents, carriers and excipients, as are well known to those skilled in the art.

In contrast to the previous view, CFH and CFHR protein have different effects, and accordingly, a combination of these two classes of proteins in a pharmaceutical composition is useful to exploit the various effects of these molecules described herein. Consequently In contrast to the teaching in WO2006 / 088950, CFH can not be replaced by CFHR, since it is alleged that they have the same effect; rather, a combination of these two classes of molecules leads to an improved effect since they show different effects on the complement system. The pharmaceutical formulations may further comprise conventional pharmaceutically acceptable adjuvants, as well known to those skilled in the art. These pharmaceutical formulations can be administered by the usual routes and include effective amounts of the active ingredients, namely CFHR functional protein optionally in combination with functional complement factor H.

Functional CFHR proteins and functional complement factor H can be obtained from natural sources such as human plasma or serum or recombinantly. The person skilled in the art is aware of methods for the isolation of natural CFHR protein or factor H protein and genetic engineering methods for the recombinant production of the functional CFHR proteins and functional factor H proteins.

Acceptable pharmaceutical carriers and excipients, as well as suitable pharmaceutical formulations, are well known, see e.g. pharmaceutical formulation development of peptides or proteins, Frokjaer et al. Taylor & Francis (2000) or Handbook of Pharmaceutical Excipients, 3rd Edition,

Kibbe et al., Pharmaceutical Press (2000).

For example, the pharmaceutical formulations may be provided as in lysed or stabilized soluble form.

The routes of administration include systemic routes of administration, such as parenteral routes of administration, e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intercerebral, intrapulmonary, intranasal or transdermal routes or enteral routes, such as oral, vaginal or rectal routes.

The therapeutically effective amount of functional CFHR protein or functional factor H protein may depend on many factors including the indication, the formulation, the route of administration, and the age and condition of the individual. The skilled person can determine the effective dose taking into account these parameters.

The pharmaceutical compositions according to the present invention may be used alone or in combination with other therapeutic agents be administered, which may optionally be incorporated into a pharmaceutical formulation.

Finally, in a further aspect, the present invention is directed to monoclonal antibodies that specifically detect CFHR protein immunohistochemically and immunochemically. In particular, these monoclonal antibodies allow a distinction between CFHR protein and CFH protein.

In particular, this monoclonal antibody is one which specifically binds to human CFHR, in particular CFHR1 protein, wherein the monoclonal antibody may have the same properties as the monoclonal antibody of hybridoma cell line JHD-7. 10. 1 deposited under the Budapest Treaty at the DSMZ, Braunschweig. This monoclonal antibody binds in the N-terminal region of the CFHR-1 protein.

Thus, in a preferred embodiment, the present invention is directed to a monoclonal antibody capable of specific binding to human CFHR protein, wherein the monoclonal antibody reacts with the same epitope of the human CFHR protein as the monoclonal antibody derived from the hybridoma cell line JHD-7. 10. 1 deposited under the Budapest Treaty at the DSMZ, Brunswick, DSM ACC 2978.

Another embodiment of the present invention relates to a hybridoma cell line expressing a monoclonal antibody of the invention.

With particular preference, this hybridoma cell line is the

Hybridoma cell line JHD-7. 10. 1 deposited under the Budapest Treaty with the DSMZ,

Brunswick, DSM ACC 2978.

These monoclonal antibodies enable methods for the specific determination of CFHR protein, in particular CFHR1 protein, in

Body fluids, such as blood, especially plasma or serum without CFH

Proteins are determined. This inventive method comprises the

Step of incubating a sample to be tested with a monoclonal antibody according to the invention and determining the monoclonal antibodies bound to CFHR, in particular CFHR1 protein. This method can be a qualitative or (semi) -quantitative method.

Such diagnostic methods are particularly suitable for determining the content of CFHR1 protein in body fluids, such as the blood, and especially the plasma, of individuals suffering from hemolytic uremic syndrome, age-related macular degeneration or on membranoproliferative glomerulonephritis and other inflammatory kidney diseases, as well as other autoimmune diseases. The deficiency of CFHR1 as well as CFHR3 in plasma represents a risk factor for the development of, for example, HUS. Therefore, an antibody according to the invention for the detection of CFHR1 in plasma is suitable for determining this risk and for detecting deficiency and, above all, adolescent patients with HUS therapeutic benefit, as the deficiency correlates with the presence of autoantibodies, which initiates a different mode of treatment for this patient, namely the reduction of the antibodies and the complementation of the CFHR proteins.

In a preferred embodiment, therefore, the present invention is directed to a method for the diagnosis of HUS comprising the steps of determining CFHR protein using the antibody of the invention in combination with the determination of autoantibodies. The diagnostic methods according to the invention may include conventional methods such as e.g. ELISA, Western blotting, immunological rapid tests, and protein-based microarrays,

In the age-related macular degeneration (AMD), the presence of CFHR1 in the plasma is also important and the development of AMD correlates with the presence of CFHR1 protein. A corresponding diagnostic procedure therefore facilitates the detection and prognosis of AMD.

Further aspects of the invention are directed to methods for the treatment of inflammatory reactions with functional CFHR proteins but also with nucleic acids coding therefor. In the following, the invention will be further illustrated by way of examples using CFHR1 protein. However, the invention is not limited to the following examples.

Procedure proteins and antibodies

Recombinant CFHR1 and deletion mutants of CFHR1 SCRS1-2 (CFHR1 / 1-2) and CFHR1 SCR3-5 (CFHR1 / 3-5) were expressed according to known methods. The proteins were expressed in Pichia pastoris and purified by means of nickel chelate affinity chromatography. Vitronectin was from BD Biosciences (Belgium). C3b, C3d, C5, C5bC6, Factor H and Factor I were obtained from Merck Biosciences (Schwalbach, Germany) and CT ₁ C8 and C9 from Comptech (Taylor, USA). Purification of natural CFHR1 CFHR1 protein was determined by hepatic chromatography (HiTrap Heparin

HP column, GE Healthcare, Munich, Germany) diluted with Sterofundin (Braun Melsungen, Germany). The proteins were eluted with a 3-step gradient of NaCl (100, 200 and 300 mM) dissolved in sterofundin. The pooled eluate fractions of 100 and 200 mM NaCl gradients were concentrated (Superdex 2000, Satohus, Germany) and the proteins were adjusted to pH 4.7 with 1X PBS. The samples were separated by SDS PAGE and the band containing CFHR1 was identified by the mobility of the size markers, the appropriate region was excised and CFHR1 was eluted, concentrated and dialysed 6x against PBS pH4.7. The mouse monoclonal antibody C18 (Alexis, Lausen, Switzerland) became the

Detection of the C-terminus of CFH and CFHR1 used. For the specific detection of CFHR1, the monoclonal antibody JHD10 from the hybridoma cell line JHD-7. 10. 1 deposited under the Budapest Treaty at DSMZ, Braunschweig, DSM ACC 2978. This antibody is directed against purified CFHR1-SCR1-2 fragment, thus binding in the N-terminal region of CFHR1. At higher concentrations of this antibody, CFHR5 and CFHR2 are also detected. Anti C5, anti C6, anti C7 were obtained from Comptech (USA).

Secondary antibodies were obtained from Dako (Glostrup, Denmark). serum samples

Normal human serum was obtained from healthy volunteers. Blood was collected from healthy volunteers and stored at -80 ⁰ C until use. Six patients with atypical HUS were also studied. Deficiency of CFHR1 was determined by Western blot analysis and verified by genetic analysis as known. CFH autoantibodies were identified by ELISA against CFH-specific antibodies as described. For CFH depletion, 150 μl of protein A Sepharose were used as matrix (GE-HealthCare, Freiburg, Germany). This was incubated overnight at 4 ° C with 300 μg / ml monoclonal antibody C18 (Alexis, USA) and 150 μg / ml monoclonal antibody B22, from own working group, incubated. The depletion of the serum was verified by Westerblot analysis. For C7 depletion, 150 μl of polyclonal goat anti-C7 (Quidel, USA) was used and depletion was performed as described for CFH. The following sera were used: human complement active

Plasma (HP), complement active HP, which were CFH removed and CFHR1 (HPΔ _C FH), complement active HP, wherein CFH were CFHR1 and C7 removed (HP _Δ CFHΔ _C7), complement active plasma of CFHR1 / CFHR3-Deffizienten healthy Persons (defHP), complement active defHP, in which CFH and C7 were removed (defHP _ΔC FHΔC7),

Cell culture and confocal microscopy

Human umbilical cord endothelial cells (HUVEC, ATCCF, CRL-1730) and retinal pigment epithelial cells (ARPE-19, ATCC CRL-2302) were cultured according to known procedures. For binding experiments, cells were incubated for 24 hours in serum-free medium by means Kurzinkubation in 0.02% Accutase (PAA, parching, Germany) and incubated at 37 ⁰ C and detached in PBS supplemented with 1% BSA. Sheep, rabbit and chicken erythrocytes were obtained from Rockland (USA). For confocal microscopy, HUVEC and ARPE-18 cells grew on coverslips (Nunc) and were washed with PBS and non-specific binding sites were blocked with PBS spiked with 1% BSA. The cells were then incubated for 60 minutes with CFHR1 or CFH (100 μg / ml) or normal human plasma (NHP, 5%). The binding of CFHR1 was performed using the monoclonal antibody JHD10 with a secondary anti-mouse antibody labeled Alexa 647 and CFH with a polyclonal antiserum specific for the N-terminal domain of CFH (anti SCR1-4) (Alexis, USA) visualized with a secondary goat anti-rabbit antibody labeled with Alexa 488. The samples were washed with 1% BSA / PBS, counterstained with DAPI and wheat germ agglutinin Texas red-595 and assayed using a laser scanning microscope LSM 510 META (Zeiss, Jena, Germany). The rabbit erythrocytes were incubated with 3b (10 μg / ml) before addition of CFHR1 (100 μg / ml), immobilized on a coverslip and stained with the monoclonal antibody JHD10. Unspecific binding of the antibodies to the cells was excluded and no signals were detected in the absence of CFHR1 or CFH. immunohistochemistry

Immunohistochemistry was performed with two human donor eyes that had no clinical documentation for early AMD and no documentation for morphological evidence of eye disease. The donor eyes were obtained at autopsy and were processed within 15 hours of death. Furthermore, normal kidney tissue was obtained from two human adult donor kidneys that were not used for transplantation. Posterior ocular preparations and parts of the decapsulated kidney were embedded in OCT compound and frozen in isopentane-cooled liquid nitrogen. Cryostat sections (6 μm) were fixed in cold acetone, blocked with 10% normal goat serum and incubated with a monoclonal mouse antibody to CFHR1 (JHD10) diluted 1: 100 in PBS overnight at 4 ° C. Antibody binding was detected using a Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, USA). Nuclear counterstaining was carried out with propidium iodide. For the preabsorption experiments, the primary antibody was treated with either CFHR1 or CFH for one hour. Binding of CFHR1 to heparin, C3b, C5 and C5bC6

MaxiSorp plastic plates (Nunc) were incubated with heparin (heparin sodium salt, Sigma, Germany) 500 units / plate or C3b, C3d (Merck Biosciences, Germany) (10 μg / ml), C5 or C5bC6 (Merck Biosciences, Germany) (5 μg / ml ) and non-specific binding sites were blocked with 2% BSA in PBS. CFHR1 (at various concentrations of 10 to 90 μg / ml) or CFH (75 μg / ml) were dissolved in binding buffer B (10 mM Na ₂ HP ₄ , 27 mM KCl, 1, 4 M NaCl, 2% BSA, pH 7, 4) was added to each plate and bound CFHR1 was incubated with monoclonal antibody C18 and bound CFH with the antiserum of SCRs 1-4 in combination with the corresponding HRP-conjugated rabbit anti-goat and anti-mouse IgG (Dako , Denmark, 1: 4000 dilution). As a control, buffer B was added directly in the absence of the primary protein. In parallel, JHD10 or mAb C18 (15 μg / ml) were incubated in a microtiter plate and used to capture CFHR1 (30 μg / ml). After washing, C5 or C5bC6 (5 μg / ml) was added in gelatin veronal buffer (Sigma, Germany) and bound proteins were identified using a monoclonal C5 antibody (Merck Biosciences, Germany). CFHR1-specific antiserum was used to confirm the binding of CFHR1 to the immobilized monoclonal antibodies. For the competition experiments, C3b (10 μg / ml) was immobilized in a microtiter plate (Nunc, Germany) and constant amounts of CFH (5 μg / ml) were added. Furthermore, increasing amounts of CFHR1 (1.3 to 26.6 μg / ml) dissolved in Buffer B were added and bound CFHR1 and CFH were detected with monoclonal antibodies JHD10 or polyclonal antiserum against SCRs 1-4 of CFH. Cofactor Assays The cofactor activity of heparin-bound CFH was measured by measuring factor I-mediated degradation of C3b by Western blot analysis. Briefly, heparin (Fluka, Buchs, Switzerland) (5 μg / ml) was immobilized in a microtiter plate (EprarEx ™, Piasso) overnight at room temperature and nonspecific binding was performed with 1% BSA in blocking buffer (20 mmol HEPES, 130 mmol NaCl, 0.05% Tween) for two hours at room temperature. For the immobilized Kompetitionsanalysen CFH was incubated with increasing amounts of CFHR1 (0.13 micrograms to 13.3 micrograms), followed by incubation at 37 ⁰ C for 15 minutes with 2 ug and 0.28 ug C3b CFI in a total volume of 50 ul. The samples were removed from the microtiter plates and incubated with sample buffer containing beta-2-mercaptoethanol and boiled for 5 minutes at 95 ^° C. The proteins were separated on a 10% SDS-PAGE, blotted onto a nitrocellulose membrane, and blotted with goat anti-human C3 (Calbiochem, 1: 1000) and HRP-conjugated rabbit anti-goat Ig (Dako, 1: 1000). developed according to general methods. The presence of the α'43 degradant of C3b was determined by densitometry. ELISA

To determine whether CFHR1 has effects on complement activation, the activity of CFHR1 was examined for each of the three complement pathways using WiELISA (Wieslab, Lund, Sweden). CFH and CFHR1 dejected NHS (1% classical and lectin and 20% alternative route) were incubated with increasing concentrations of CFHR1 (20 to 80 μg / ml) for 10 minutes on ice. Equal amounts of DPBS were added to each sample to eliminate buffering effects. After incubation, the samples were treated according to the manufacturer's instructions. To study the CFHR1 regulation of C3 convertase, C3 convertase was determined by incubation of C3b (2 μg / ml) and C3 (80 μg / ml) with factor D (4 μg / ml) and factor B (40 μg / ml) in activation buffer (20 mM Hepes, 144 mM NaCl, 7 mM MgCl ₂ , 10 mM EGTA, pH 7.4). The activity of C3 convertase was determined after incubation of constant amounts of C3 (18 μg / ml) and increasing amounts of CFHR1 (25 and 50 μg / ml) or CFH (50 μg / ml) or 25 μg / ml human serum albumin (HSA ) determined by C3a formation. C3a concentrations were determined by ELISA (Quidel, USA) according to the manufacturer's instructions. Erythrocyte lysing assay Sheep erythrocytes were incubated with 30% v / v CFH and CFHR1 deputed human plasma in AP buffer (20 mM Hepes, 144 mM NaCl, 7 mM MgCl ₂ , 10 mM EGTA, pH 7.4). The deputed plasma was tested for hemolysis of the sheep erythrocytes prior to the experiment. Hemolytic experiments were tested in HEPES / EGTA buffer (20 mM Hepes, 144 mM NaCl, 7 mM MgCl ₂ , 10 mM EGTA, pH 7.4). Increasing concentrations of CFHR1 (5-160 ug / ml) were added to the plasma and incubated for 10 ⁷ sheep red blood cells at 37 ⁰ C for 15 minutes with about 2 x. After incubation, the mixture was clarified by centrifugation and the absorbance was measured at 415 nm in the supernatant. Furthermore, dejected plasma samples were incubated with equal amounts of CFH, vitronectin or BSA. In one experiment, the formation of complement activation products C3a and C5a was determined by ELISA. An aliquot of each sample was taken from the hemolysis assay and immediately diluted in ice-cold Hepes / EGTA buffer (C3a: 1: 4000, C5a: 1: 100) and stored on ice. The C3a and C5a concentrations were measured with commercially available ELISAs according to the manufacturer's instructions (C3a: Quidel, USA, C5a: DRG Diagnostics, Marburg, Germany).

HP deficient in CFHR1 and CFHR3 was depleted by the complement component C7 to inhibit formation of the terminal membrane attacking complex (MAC). Polyclonal C7 antiserum (Comtech, USA) was coupled in 1 ml of protein A Sepharose column and incubated with CFHR1 and CFHR3 deficient plasma. To this depletion serum, 25 μg / ml or 50 μg / ml recombinant CFHR1 was added and incubated with 2 × 10 ⁷ rabbit erythrocytes in Hepes / EGTA buffer for a period of 5 to 30 minutes to activate the alternative complement pathway. Every 2.5 minutes, aliquots were taken from this activated plasma, the complement activation was with the help of a

Protease inhibitors (Complete, Roche, Germany) stopped and on the hemolytic activity by incubation with a small amount of deficient HP (1

%) as a source of C7-C9 and 2 x 10 ⁷ chicken erythrocytes. Hemolysis was determined for each activation time by measuring absorbance at 415 nm.

Hemolysis of chicken erythrocytes was determined in CFHR1 / CFHR3 deficiencies,

Complement inactivated (20 mM Hepes, 144 mM NaCl, 10 mM EDTA, pH 7.4) HP with constant concentrations of C5b6 (5 ng / ml) protein complexes and increasing amounts of CFHR1 (25 to 100 μg / ml). The deficient plasma was incubated with C5b6 and CFHR1 for 15 minutes at 37 ⁰ C, and the lysis of chicken erythrocytes was determined by assaying the supernatant at 415 nm. To demonstrate the specificity of the CFHR1 function, deficient serum was incubated in parallel with equal amounts of CFH or BSA in the presence of C5b6. Plasma-derived CFHR1 activity was examined in the same hemolytic assay. C5b6 complexes (5 ng / ml) were mixed with either plasma-derived CFHR1 (0.75 μg / ml) or recombinant CFHR1 (5 μg / ml) in 20 mM Hepes, 144 mM NaCl, 10 mM EDTA, pH 7.4 pre-incubated for 5 minutes at 20 ⁰ C. Sheep red blood cells (2 x 10 ⁷⁾ were added and after 10 minutes at 20 ⁰ C, the terminal moieties C7 (final concentration 1 ug / ml), and C9 added to C8 (0.2 ug / ml) (1 ug / ml). After incubation for 30 minutes at 37 ^° C., the release of hemoglobin at 415 nm was measured. Similarly, the effect of the terminal pathway inhibitor, vitronectin (1.25 μg / ml) and BSA (1.25 μg / ml) was investigated. Flow cytometry Flow cytometry was used to examine the effect of CFHR1 on C5 deposition on erythrocytes during complement activation. In order to allow complement activation, but to prevent hemolysis, CFHR1 / CFHR3 deficient plasma was treated by immunoaffinity chromatography to remove CFH and C7 (defHP _ΔCFHΔC7 ). For each time point sheep and rabbit erythrocytes in 30% v / v defHP _Δ c _FHΔC7 in attendance or absence of 50 ug / ml were incubated CFHR1. To prevent too rapid degradation of the C3 / C5 convertase complex, sodium chloride in the buffer was replaced with mannitol (20 mM Hepes, 250 mM mannitol, 8 mM MgCl ₂ , 10 mM EGTA, pH 7.4). At any time, the sample was frozen in ice modified buffer with 1% w / v BSA. To inhibit complement activation, the buffer contained a protease inhibitor mixture (Complete Inhibitor Mix, Roche, Germany). C5 was detected using a mouse monoclonal anti-C5 antibody (Quidel, USA). The erythrocytes were measured by means of a B & D LSR II with suitable laser and filter settings. 50000 events were routinely counted. Binding of serum-derived CFHR1 to HUVEC cells was assayed by incubating the HUVEC cells, which were kept serum-free for 3 days, in 25% normal human serum for 30 minutes. The cells were washed and binding of CFHR1 was determined with the monoclonal antibody JHD10. Coating surfaces with CFHR1

CFHR1 can be used either as a recombinant protein or after purification from plasma on surfaces according to standard procedures, e.g. in ELISA approaches. For this purpose, CFHR1 is incubated with the material in a buffer solution and then washed intensively with a physiological buffer. Alternatively, directed immobilization via monoclonal antibodies, e.g. the CFHR1 specific mAB JHD 10 or also C18 or polyclonal ancesters are possible. Another way of attachment is to coat the surface with CFHR1 ligands, e.g. with heparin. Since CFHR1 binds to heparin and other ligands, it is possible to coat the surface with CFHR1. Statistical analysis

The statistics were carried out with the help of the Student T-Test. P values less than 0.05 were considered significant.

Results

The experiments showed that CFHR1 binds to C3b, C3d, heparin on human cells as shown in FIG.

CFHR1 uses the C-terminus for both C3b and heparin binding since only the SCRs3-5 deletion mutants bind the immobilized C3b and heparin to the SCRsI-2 deletion mutants. Furthermore, it was shown that CFHR1 binds to cell surfaces, see Figures 1d to e.

In FIG. 2, CFHR1 staining is shown on tissue sections of kidney and retinal tissue and hereby the new monoclonal antibody disclosed herein was obtained Antibody JHD10 is used, which recognizes an N-terminal epitope of CFHR1 protein, see Figure 2. The specificity of the antibody is set forth in FIG.

CFHR1 competes with CFH for the same binding sites. As shown in Figure 4, the two molecules compete for binding to C3b and CFHR1 can replace CFH and reduce CFH-mediated activity.

In Figure 4, the characteristic of CFHR1 is set out to regulate the activation of the alternative complement pathway. CFHR1 and the known complement inhibitor vitronectin showed similar inhibitory effects. Unlike CFH. An effect on the classic or the Lectinweg, however, did not show. CFHR1 does not affect C3 convertase, see Figure 5. However, CFHR1 has a dose-dependent inhibitory effect on complement-mediated lysis. That is, CFHR1 has an impact on the formation and activity of the MAC.

As shown in Figure 6, the CFHR1 regulates the C5 convertase of the alternative path. The molecule has an inhibitory effect on the formation of C5b, but not on C3b, and may also inhibit C5a generation in a dose-dependent manner, see Figure 6a.

Figure 7 shows that CFHR1 binds to C5 and C5b6 with the N-terminal region of the molecule. CFHR1 prevents the formation of the MAC, see Figure 7c. To confirm that this effect can not be attributed to recombinant artifacts, native purified

CFHR1 protein used to confirm the effects, see Figure 7d.

INFORMATION ON A MICROORGANISM LAYING DOWN OR OF BIOLOGICAL MATERIALS

(Rule \ 3bis PCT)

A. The following information relates to the deposited micro-organism or other biological material mentioned in the description on page] β, lines 15-23

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Name of the depository DSMZ

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Inhoffenstr. 7B 38124 Brunswick

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September 23, 2008 DSM ACC2978

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Authorized Agent Agent Agent Wallentin, Marko

Form PCT / RO / 134 (July 1998, reprinted January 2004)

Claims

claims

Use of a CFHR molecule, in particular a CFHR protein, or a functional fragment or functional derivative thereof, which inhibits the enzyme C5 convertase, for the treatment or prophylaxis of autoimmune diseases and inflammatory reactions.

2. Use according to claim 1, wherein the inflammatory response is an inflammatory response in the context of an autoimmune disease.

Use according to claim 1 or 2, wherein the inflammatory response is a reaction induced in the context of sepsis, rheumatoid arthritis, Alzheimer's disease, atherosclerosis, lupus erythematosus or antiphospholipid syndrome.

4. Use according to any one of the preceding claims, wherein the CFHR protein or a functional fragment or a functional derivative thereof prevents the formation of the terminal membrane-attacking complex.

5. Use according to one of the preceding claims, wherein the alternative way of complement activation influenced, in particular inhibited.

6. Use of a CFHR protein or a functional derivative or a functional fragment thereof for inactivating complement activation, in particular in transplantation or dialysis.

7. Use of CFHR protein or a functional fragment or a functional derivative thereof for coating implants and surfaces which may come into contact with body fluids.

Use according to any one of the preceding claims, wherein the CFHR protein is the CFHR1 protein.

9. Coating for devices which come into contact with body fluids, in particular plasma or blood, characterized in that it has on the surface CFHR protein, functional fragment or functional derivative thereof.

A coating according to claim 9, characterized in that it is one for an implantable device, said implantable device being intended for use in the body of an individual.

11. Device which comes into contact with body fluids and in particular blood, in particular implantable device, characterized in that on the surface of CFHR protein, functional derivative or functional fragment thereof has been applied.

12. A pharmaceutical composition comprising functional CFHR protein in combination with functional factor H and optionally with other pharmaceutically acceptable diluents, carriers and excipients.

13. A pharmaceutical composition according to claim 12 comprising functional CFHR1 protein.

Monoclonal antibody which specifically detects a CFHR protein immunohistochemically and immuno-biochemically and is capable of specific binding to human CFHR, in particular CFHR1 protein, wherein the monoclonal antibody has the same properties as the monoclonal antibody

Antibody of the hybridoma cell line JHD-7. 10. 1 deposited under the Budapest Treaty at DSMZ, Braunschweig, Germany, DSM ACC 2978.

The monoclonal antibody of claim 14 which is capable of specific binding to human CFHR protein, wherein the monoclonal antibody reacts with the same epitope of the human CFHR protein as the monoclonal antibody derived from the hybridoma cell line JHD-7. 10. 1 deposited under DSMZ ₁ Braunschweig, DSM ACC 2978, according to the Budapest Treaty.

16. A monoclonal antibody according to any one of claims 14 or 15, characterized in that it does not react with the complement factor H.

17. A hybridoma cell line expressing a monoclonal antibody according to any one of claims 14 to 16.

18. A method for the determination of CFHR molecules, in particular of CFHR1 protein, in body fluids, comprising the step of incubating a sample to be tested with a monoclonal antibody according to any one of claims 14 to 16 and determination of antibody-bound CFHR molecules.

The method of claim 18 for determining CFHR1 content in a body fluid of an individual for the diagnosis of hemolytic uremic syndrome, age-related macular degeneration, or membranous proliferative glomerulonephritis and other inflammatory kidney diseases.

20. The method of claim 20 for the diagnosis of hemolytic uremic syndrome further comprising the step of determining autoantibodies in a body fluid of the subject.

21. The method according to any one of claims 18 to 20, wherein the body fluid is blood, plasma or serum.