WO2012065852A2

WO2012065852A2 - Therapeutic protein

Info

Publication number: WO2012065852A2
Application number: PCT/EP2011/069326
Authority: WO
Inventors: Meinrad Gawaz; Iris Irmgard Mueller; Christoph Leder; Christina Filzmayer
Original assignee: Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum
Priority date: 2010-11-18
Filing date: 2011-11-03
Publication date: 2012-05-24
Also published as: WO2012065852A3; DE102010052941A1

Abstract

The invention relates to the use of the protein gremlin and a fusion protein, containing a) a first polypeptide which has an immunoglobulin Fc domain, and b) a second polypeptide which has gremlin or fragments or variants thereof which possess the BMP binding function of gremlin, wherein the first polypeptide and the second peptide are linked to one another directly or via linker peptide. The invention further relates to the use of the fusion protein treating diseases.

Description

Therapeutic protein

The present invention relates to a protein having a therapeutic, in particular anti-inflammatory or immune modulating potential, as well as its use for the treatment of diseases in which inflammatory, immunomodulatory and / or generative processes play a role, in particular in the cardiovascular area in the Atherosclerosis, after acute myocardial infarction, in the context of acute and chronic myocarditis, and in advanced heart failure, as well as inflammatory autoimmune diseases; The invention further relates to a fusion protein containing the protein, a nucleic acid molecule coding therefor, a pharmaceutical composition containing the protein, and the use of the protein or the pharmaceutical composition for therapeutic treatment. Illnesses in which inflammatory, immunomodulatory and / or generative processes play a role, in particular in the cardiovascular area in atherosclerosis, after acute myocardial infarction, in the context of acute and chronic myocarditis and in advanced heart failure, and inflammatory lung and autoimmune diseases, are still in their genesis not or not fully explored and often represent highly complex processes in which many factors are often involved.

Thus, for example, atherosclerosis is a highly complex active pathological process, in the center of which there is an inflammatory reaction in the walls of the blood-carrying vessels of an affected individual. The development of atherosclerosis, the so-called atherogenesis, can be divided into several phases.

The early phase of atherogenesis is characterized by a so-called endothelial dysfunction. A number of different risk factors, such as Smoking, obesity, physical inactivity, hyperlipoproteinemia and type I diabetes as well as other factors not yet identified lead to endothelial damage. The permeability of the endothelium for lipoproteins and other circulating substances in the plasma increases as a result. As a result, endothelial cells are activated and increasingly express so-called adhesion molecules on the cell surface. Above all, so-called selectins initially mediate the temporary contact of certain blood cells such as monocytes and T lymphocytes with the endothelium. Another group of adhesion molecules, the so-called Cellular Adhesion Molecules (CAMs), leads to the firm attachment of these cells to the vessel wall.

In the further course, especially monocytes and, to a lesser extent, T lymphocytes enter the subintimal space. This invasion is mediated by another set of molecules, such as the chemokine monocyte. Chemoattractant protein-1 (MCP-1) is mediated. It comes to the differentiation of monocytes in macrophages in the subintimal space.

In the damaged endothelium, there is also the oxidation of low-density lipoprotein (LDL) and thereby the formation of oxLDL. The oxLDL is released into the subintimal space, where it loads the macrophages produced there from monocytes. These macrophages are transformed by the loading with oxLDL into so-called foam cells, the characteristic cells of the atherosclerotic plaques, which as further constituents i.a. T lymphocytes and from the media immigrated smooth muscle cells.

These plaques deposit on the arterial walls and are thereby covered by a stabilizing fibrous cap consisting of smooth muscle cells and extracellular matrix. Atherosclerotic plaque is now at the center of an inflammatory reaction in which various inflammatory mediators are produced, such as cytokines, chemokines, proteases, etc. This can lead to tissue necrosis, in the vicinity of which lime salts are deposited. As a result, the vessel lumen can be narrowed to complete closure with the result of circulatory disorders.

Also, in other inflammatory diseases, such as. The cardiovascular system, the lung, or in inflammatory autoimmune diseases in general play, and this has been the research in recent years, including. Cytokines also have a specific, if not even causal, role.

One approach to the treatment of cytokine-triggered, caused or induced diseases would therefore be to specifically target the release / formation or action of certain cytokines.

The object of the present invention is to provide such a novel agent. This object is achieved by a protein having the sequence of Gremiin, or variants or fragments thereof having the BMP binding function of Gremiin, for the treatment of diseases associated with a reduction in MIF formation and / or the CXCLl formation and / or MCP-1 formation and / or foam cell formation are treatable.

This object is further achieved by a fusion protein which is (a) a first polypeptide having an immunoglobulin Fc domain, and (b) a second polypeptide selected from Gremiin or variants or fragments thereof containing the Possess BMP (Bone Morphogenetic Proteins) binding function of Gremiin. It is particularly preferred if the fusion protein towards the N-terminus to the C-terminus (a) a first polypeptide having an immunoglobulin Fc domain, and (b) a second polypeptide selected from Gremiin or variants or fragments thereof containing the BMP (Bone Morphogenetic Proteins) binding function of Gremiin.

The object underlying the invention is further solved by a pharmaceutical composition comprising the protein having the sequence of Gremiin, or variants or fragments thereof, which have the BMP binding function of Gremiin, or the fusion protein according to the invention in a contains pharmaceutically effective amount, optionally in combination with a pharmaceutically acceptable carrier, and by the use of the inventive fusion protein or the pharmaceutical composition containing it as a medicament, in particular for the treatment of atherosclerosis, ischemic heart disease, myocardial infarction, stroke, heart failure, myocarditis, inflammatory cardiomyopathy, inflammatory rheumatic and autoimmune diseases, for the regeneration of ischemic tissue after mycardial infarction, and in ischemia / reperfusion after organ transplantation.

The object underlying the invention is completely solved in this way. The protein Gremiin, also known as "Drm", belongs as a protein of the DAN family to the BMP (bone morphogenetic proteins) antagonists and plays a major role in differentiation processes of various organ systems during the embryonic Development. Thus, for example, Gremini-1 is known to undergo and inhibit BMP-2, -4 and -7 antagonistic reactions.

BMPs also control numerous physiological and pathological processes in the adult organism. The BMPs are a group of similar signaling proteins that can be used to affect adjacent cells and thus constitute cytokines. regulate the expression of numerous target genes, thereby influencing molecular mechanisms that are important for the physiological function of monocytes / macrophages, endothelial cells and smooth muscle cells. BMPs influence u.a. the adhesion behavior of endothelial cells and smooth muscle cells, as well as the activity of metalloproteinases in different cell types. The role of BMPs in the genesis and progression of atherogenesis has been poorly understood, and little is known about the role of their antagonists.

The present inventors have now been able to show that Gremiin secretes the secretion or expression of the macrophage migration inhibitory factor, the chemokine CXCL1 and the monocyte chemoattractant protein-1 (monocyte chemoattractant protein; MCP-1) lowers. Gremiin inhibits foam cell formation, regulates monocyte migration, and inhibits the activity of matrix metalloproteinase 2 (MMP2). Thus, the occurring at the artherogenesis foam cell formation can be successfully prevented and thus the development of atherosclerosis can be successfully controlled. By binding of Gremiin to the BMPs - and thereby by the inhibition of BMPs - their influence on, inter alia, monocytes for foam cell formation in the artherogenesis can be suppressed, and inhibits the formation / secretion of the "Macrophage Inhibitory Factor" (macrophage inhibition factor) become. The macrophage migration inhibitory factor (MIF) was first introduced in 1966 as a cytokine secreted by T-lymphocytes with the ability to inhibit the undirected migration of macrophages. Meanwhile, MIF has been detected in many organ systems, and it is believed that this cytokine is also involved in cell-mediated immunity, immune regulation, and inflammatory responses, such as in rheumatoid arthritis. In addition, MIF promotes plaque formation and plaque growth, promotes matrix metalloproteinase 2 and 9 activity, and regulates the adhesion and migration of monocytes and macrophages. MIF enhances myocardial damage after ischemia and stimulates myocardial apoptosis.

The inhibition of MIF by means of Gremiin therefore provides an effective tool for controlling diseases in which MIF, but also MCP-1 and CXCL1, play a triggering or important role.

The protein Gremiin used for inhibiting MIF and / or CXCL1 and / or MCP-1 can have the sequences SEQ ID No. 2 (murine) or 3 (human) listed in the attached sequence listing, as well as other cross-reactive ones Species, or fragments or variants thereof, which still possess the same binding property as Gremiin.

The term "fragment" or a "variant" which has the binding function of the respective polypeptide is understood herein to mean an amino acid sequence which differs from the wild-type sequence or the sequence given herein by one or more amino acid substitutions , Such modified amino acids may have "conservative" amino acid substitutions in which the exchanged amino acid has the same or similar properties as the replaced amino acid. Similar small changes may also include amino acid deletions and / or insertions. Guidance for determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may, for example, be found using computer programs known in the art become. The protein or polypeptide variants or fragments thereof encompassed herein include, in particular, Gremlin proteins and variants derived therefrom, both murine and human, or fragments thereof, each still having the Gremlin binding property, identical or substantially equivalent. This means that within the scope of the present invention, Gremlin fragments or variations are suitable which, like Gremiin, still possess a binding property to BMPs. For example, whether a modified Gremlin polypeptide possesses the binding properties of the unmodified Gremlin polypeptide can be assayed in in vitro assays, as described further herein below. Thus, variants also include polypeptides each having approximately 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the respective wild-type polypeptide / protein , In order to determine the percentage of the sequence identity of two amino acid or nucleic acid sequences, it is possible to proceed according to methods known in the prior art, for example by means of an alignment and calculation by means of a mathematical algorithm.

The diseases or diseases for the treatment of the use of the protein Gremiin, or of variants / fragments thereof, is suitable, include all inflammatory and immunomodulatory diseases, u.a. Atherosclerosis and ischemic heart disease, myocardial infarction, stroke, heart failure, myocarditis of inflammatory cadriomyopathy, inflammatory rheumatic and autoimmune diseases, for the regeneration of ischemic tissue after mycardial infarction, and in ischemia / reperfusion after organ transplantation. All of these diseases have in common that they have shown or derived the involvement of MIF, CXCL1 and MCP-1.

As already mentioned above, the invention also relates to a fusion protein comprising fusion protein a) a first polypeptide having an immunoglobulin Fc domain, and b) a second polypeptide selected from Gremiin or variants or fragments thereof, that possess the BMP binding function of Gremiin. The first polypeptide is designed in terms of its amino acid sequence such that it has the Fc domain of an immunoglobulin; the Fc domain

The second polypeptide selected from Gremiin or variants or fragments thereof having the BMP binding function of Gremiin is thereby designed by suitable choice of the amino acid sequence such that it binds to BMP. For the purposes of the present invention, both murine Gremiin and human Gremiin as well as other human cross-reactive species are suitable for realizing the fusion protein.

In the present context, a "fusion protein" is understood as meaning a hybrid protein or an artificial protein which can be produced in vitro as well as in vivo by molecular biological or chemical methods known in the art. Thus, for example, the fusion protein can be produced by conjugation of two (or more) polypeptides by means of one or more chemical reagents or by recombinant DNA technologies, ie by genetic "linking" of the nucleic acids coding for the proteins. In this case, it is possible to generate the fusion protein by using customary expression vectors which code for the fusion protein according to the invention. These expression vectors are introduced into a suitable cell, which then produces the fusion protein.

The terms "linker" or "linker peptide", which are used interchangeably herein, in the present invention, an amino acid or a nucleic acid sequence encoding it with up to 100 amino acids / bases understood, which serves to link two functional polypeptides, and the optionally generates a distance between the two functional polypeptides, without possessing a binding property to other molecules. This is therefore a "neutral" sequence, which can fulfill or fulfill no other than the functions just described. In certain embodiments For example, the linker peptide may also have cleavage sites for certain enzymes via which the two polypeptides can be separated from one another.

By "binding property of Gremiin" is meant the ability of Gremiin (or fragments and derivatives derived therefrom) to bind to BMPs, in particular to BMP-2, -4 or -7. By binding of Gremiin to the BMPs, the latter, as already mentioned, are inhibited.

It is preferred if the second polypeptide has a human Fc domain of an immunoglobulin, in particular of IgG ₂ .

By linking Gremiin with the Fc portion of an immunoglobulin, the purification of the fusion protein is greatly facilitated, and the serum stability thereof is increased. As a relatively small molecule, Gremiin has only a short serum half-life, which limits its clinical use. By linking to Fe, the stability of Gremiin in vivo can be improved.

"Fe" stands for "fragment crystallizable"; this fragment is formed by papain cleavage of the IgG molecule next to the two Fab fragments. The Fc domain consists of the paired C _H 2 and C _H 3 domains, including the hinge region, and contains the part of the immunoglobulin that is responsible, inter alia, for the dimerization reaction. Advantageously, commercially available human or mouse Fc DNA can be used, which can either be isolated from commercially available cDNA libraries by PCR, or which is already cloned into plasmids, which in turn can be purchased (for example, available from the company Invitrogen, San Diego, USA).

In one embodiment, it is preferred if a variant of the Fc domain or a synthetic Fc fragment is used, which in the complex ment and Fc receptor binding region is mutated such that activation of the immune system is largely reduced and possibly even absent.

This measure has the advantage that the compatibility of the fusion protein according to the invention is further increased. Thus, according to findings of the inventors for the inventive function of the fusion protein namely, if the second polypeptide has only the stabilizing function of the Fc domain of the antibody, but not the effector functions that cause activation of the immune system. For this reason, it is also possible, for example, to use a synthetic Fc fragment which has been mutated in the complement and Fc receptor binding region in such a way that activation of the immune system is largely reduced and may even be absent.

In particular, it is preferred if the first polypeptide has an amino acid sequence with SEQ ID NO: 1 from the attached sequence listing showing the sequence of the IgG ₂ Fc moiety.

With this measure, a polypeptide is advantageously provided, which is derived from the Fc fragment of the human IgG ₂ - variant.

In another embodiment, the second polypeptide has an amino acid sequence selected from SEQ ID NO. 2 or 3, or variants or fragments thereof that have the BMP binding function.

SEQ ID NO: 2 represents the amino acid sequence of murine Gremiin, SEQ ID NO: 3 the amino acid sequence of human Gremiin, in both cases without the signal sequence or the signal peptide comprising 24 amino acids.

For a person skilled in the art, it is understood that the fusion protein does not necessarily fulfill the full function in order to fulfill the function according to the invention. must have identical amino acid sequence of Gremiin. Rather, the inventive function of the fusion protein is also fulfilled if the second polypeptide a fragment or a sequence variant of Gremiin, but still exerts the BMP binding function of Gremiin in possibly attenuated form. It is known that the proteinogenic amino acids are divided into four groups, namely polar, non-polar, acidic and basic amino acids. The exchange of a polar amino acid for another polar amino acid, for example glycine for serine, generally leads to no or only a slight change in the biological activity of the corresponding protein, so that such an amino acid exchange leaves the fusion protein according to the invention largely unaffected in its function. Against this background, the present invention also encompasses such a fusion protein which as a second polypeptide is a variant of Gremiin in which one or more amino acids of one of the said amino acid classes is exchanged for another amino acid of the same class. Such a sequence variant is preferably about 70%, more preferably about 80% and most preferably about 90 to 95% homologous to the amino acid sequence of Gremiin.

In further embodiments, it is preferred if the first polypeptide is linked directly or via one or more linker peptides with the second polypeptide. In one embodiment, it is preferable if the linker peptide contains the amino acid sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-GlyGly-Gly-Ser (SEQ ID No. 4). This sequence acts as a kind of spacer. It is understood that another sequence can also be used, preferably a sequence which is similar in polarity to the aforementioned. It is within the skill and skill of the art to identify suitable sequences and incorporate them into the fusion protein to link the first peptide to the second polypeptide.

According to a further embodiment, the linker peptide via which the first polypeptide is linked to the second polypeptide in a preferred embodiment, optionally in addition to the mentioned in the previous paragraph SEQ ID NO. 4 also an amino acid sequence with SEQ ID NO. 5 from the attached sequence listing. This amino acid sequence represents a thrombin site. This site can be inserted to solubilize Gremiin from Fe, if desired, by thrombin digestion. It is understood that other linker peptides can be used to link the two polypeptides, if desired, in particular those that lead to no or only a slight change in the biological activity of the corresponding fusion protein compared to the indicated linker molecule, so that such an amino acid exchange the fusion protein according to the invention is largely unaffected in its function. It is understood, however, that the insertion of such a linker peptide is not mandatory, and this can be completely dispensed with.

According to one embodiment of the fusion protein according to the invention is preferred if it has the amino acid sequence with the SEQ ID Nos. 7 or 8. The two sequences differ in that the sequence designated by SEQ ID No. 7 has a sequence for the secretion signal, whereas the sequence with SEQ ID No. 8 does not include this sequence. Furthermore, instead of the murine Gremlin sequences given in the sequences 7 and 8, the human Gremlin sequence can also be used; this is shown in SEQ ID NO. 3, so that a fusion protein is likewise protected which has the SEQ ID NO. 9 owns.

The invention further relates to a nucleic acid molecule comprising a sequence selected from the group: a) the nucleic acid sequence which codes for the fusion protein with the SEQ ID Nos. Or 8 or 9, or a variant thereof, which for the same polypeptide according to the degeneracy of the genetic code; b) a nucleic acid sequence coding for a polypeptide which codes for at least 70% sequence homology to the polypeptide by the SEQ ID NOs. ID Nos. 7, 8 or 9, wherein the nucleic acid sequence encodes a polypeptide comprising from its N-terminus to its C-terminus IgG2 Fe, a nucleic acid sequence encoding a first linker epitope, a nucleic acid sequence encoding a thrombin site, one for Gremiin or a nucleic acid sequence encoding a functional variant thereof which is capable of binding to BMP; c) a nucleic acid encoding a polypeptide having in the 5'-3 'direction a first portion encoding IgG2 Fe, a second portion representing the amino acid sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly -Gly-Gly-Ser-Gly-GlyGly-Gly-Ser, a third section encoding a thrombin site, and a fourth section encoding gremiin or a fragment or variant thereof encoding the BMP binding function of Gremiin has.

The invention further relates to a vector comprising a nucleic acid according to the invention; Furthermore, the invention relates to a fusion protein encoded by a nucleic acid according to the invention, as well as a cell expressing the fusion protein according to the invention.

The fusion proteins of the invention can be prepared using recombinant expression vectors known in the art. In the present case, a "vector" / "expression vector" is understood as meaning a replicable DNA construct which is used for expression of the DNA encoding the fusion protein according to the invention; it comprises a transcription unit comprising an array of one or more genetic elements having a regulatory role in gene expression, for example promoters, operators or enhancers, operatively linked to a DNA sequence encoding the fusion protein of the invention expressed in mRNA transcribed and translated into the protein, and a suitable transcriptional and translational onstart and translation stop sequences. The choice of promoter and other regulatory elements will generally vary depending on the (host) cell used. In a preferred embodiment, the nucleic acid encoding the fusion protein of the invention is transfected into a host cell using recombinant DNA techniques; Suitable host cells include prokaryotic, yeast or eukaryotic cells, and will be readily apparent to one of ordinary skill in the art, in light of the present specification.

For the production of the recombinant fusion protein, the host cells which have been transfected or transformed with an expression vector carrying the nucleic acid encoding the fusion protein according to the invention are cultured under conditions which promote the expression of the fusion protein according to the invention. The fusion protein can then be purified and isolated from the culture medium or host cells by methods known in the art (see, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3rd Edition).

Accordingly, the invention also relates to cells which contain the nucleotide sequences coding for said peptide, preferably in an expression vector, and in which the polypeptide is expressed therewith. For this purpose, the polynucleotides / nucleic acid sequences can be functionally linked to suitable expression control sequences in order to produce the relevant peptide recombinantly. Many suitable expression control sequences are known in the art. General methods for expression of recombinant proteins are also known and exemplified by Kaufman in Methods in Enzymology (1990), 185: 537-566.

Various cell types can serve as host cells for the expression of the proteins / peptides according to the invention, by way of example suitable mammalian host cells are mentioned here, such as, for example, monkey COS cells, ovarian cells of Hamsters (CHO cells), human kidney 293 cells, human epidermal 431 cells, human Colo205 cells, CV-1 cells, other transformed primate cell lines, cell lines derived from in vitro cultures of primary tissue or primary explants, Etc.

Also suitable are insect expression systems. Here, the polynucleotides / nucleic acid sequences of the invention are linked to control sequences in insect expression vectors. These techniques are also known in the art. Alternatively, the proteins / peptides may also be produced in lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. By way of example, Saccharomyces ssp. and Escherichia coli, which can express heterologous proteins.

To isolate the proteins expressed in the exemplified cells, the appropriately transformed cells are cultured under suitable conditions in order to express the protein / peptide. Subsequently, the protein can be isolated from culture supernatants or from cell extracts and purified by methods known in the art.

According to a further embodiment, the invention also relates to a pharmaceutical composition containing a fusion protein of the invention in a pharmaceutically effective amount, optionally together with a pharmaceutically acceptable carrier, diluent or excipient, and / or optionally with other pharmaceutically active substances.

Pharmaceutically acceptable carriers, optionally with further additives, are well known in the art and are described, for example, in the paper by Kibbe A., Handbook of Pharmaceutical Excipients, Third Edition, American Pharmaceutical Association and Pharmaceutical Press 2000. According to the invention, additives include any compound or composition which is advantageous for a therapeutic use of the composition, among which salts, Binders, solvents, dispersants, and other substances commonly used in the formulation of drugs.

The fusion protein according to the invention can be integrated into a suitable administration for the respective therapy. Examples of administration include parenteral, e.g. intravenous, intradermal, subcutaneous, transdermal, transmucosal administration. With a composition prepared according to the invention, which contains the fusion protein according to the invention, and which is administered to the patient to be treated, for example injected, the fusion protein accumulates via gremiin in the area of the lesions and can bind to BMPs, which in turn inhibit them. As a result, it is possible, for example, to successfully prevent foam cell formation in atherosclerosis, or to block inflammatory reactions which are triggered by BMPs. This provides a highly effective tool for the treatment of diseases such as atherosclerosis, rheumatoid arthritis and multiple sclerosis.

It is understood that in the present invention, not only the above-described fusion protein is suitable for use in the treatment of atherosclerosis or inflammatory autoimmune diseases, but also unfused Gremiin itself. Therefore, the present invention also relates to the use of Gremiin itself for the treatment of said diseases.

Alternatively, and according to another embodiment, the protein Gremiin may be fused with other polypeptides which stabilize the protein, such as Fe, and may be cleaved again by Gremiin in vivo. In another embodiment, Gremiin can be modified, for example, with polyethylene glycol and thereby stabilized (PEGylation), and thereby also used in this form for the treatment of the listed diseases. It is understood that the above-mentioned and below to be explained features can be used not only in the combination given, but also in other combinations or alone, without departing from the scope of the present invention.

Description of preferred embodiments

Brief description of the figures

Further advantages will become apparent from the accompanying description of the examples and the accompanying figures. Show it:

1 shows the analysis of Gremlin expression in various cell types of the human arterial vessel wall; A: RT-PCR for the semiquantitative presentation of the expression of gremiin at the RNA level; B and C: immunohistochemical staining for the expression of gremiin at the protein level;

Fig. 2 foam cell formation under the influence of Gremiin; A: foam cell formation under the influence of 500, 1000, and 2000 ng / ml medium; B: foam cell formation under the influence of Gremiin 1000 ng / ml and PRDC, BMP-2, -4 and -7 and 1000 ng / ml each in 5 independent foam cell cultures;

FIG. 3A: Analysis of the supernatants from the foam cell cultures in the cytokine array; FIG. B, C, D: concentrations of MIF, MCP-1 and CXCL1 under the influence of gremiin in the supernatant of monocytes, macrophages and foam cells; Fig. 4 is a graph showing the reduction in MIF concentration when Gremiin is given already after 8 hours incubation of monocytes;

Fig. 5 is a graph showing the apoptosis rate results under the influence of gremiin in macrophages / foam cells compared to PRDC and BMP2;

Fig. 6 shows the results of experiments to study migration inhibition of monocytes by Gremiin in the Boyden chamber (A); and (B) and (C): the results of studies on the regulation of MMP2 activity by Gremiin;

Fig. 7 shows the schematic structure of murine Gremlin-Fc (upper part) and the corresponding amino acid sequence (lower part);

8 shows the detection of the purified proteins by means of Western blot analysis;

and

Figure 9 shows the detection of binding of murine Gremlin-Fc to BMP-4. Examples

In preliminary experiments, the expression of Gremiin in various cell types of the human arterial vessel wall were examined. For this purpose, pieces of tissue from arterial vessel walls of various patients who had to undergo vascular surgery, intraoperatively obtained and then isolated endothelial cells and smooth muscle cells from it. Monocytes were isolated from the peripheral blood of healthy adults. CD34 + adult stem cells were obtained from umbilical cord blood. The results for the expression analyzes are shown in FIG. 1: FIG. 1A shows the results of the RT-PCR (reverse transcriptase polymerase chain reaction): For this purpose, the RNA was isolated from the individual cell types by means of the RNeasy Mini Kit from Qiagen (Hilden, Germany) purified and used to carry out the RT-PCR. The primers used were "exon-spanning" to prevent false positive results from contamination with genomic DNA. As can be seen from Fig. 1A, Gremiin showed the highest expression in smooth muscle cells; furthermore, its expression could be detected in monocytes, macrophages and foam cells. The macrophages and foam cells were formed from the isolated monocytes by culturing in serum-rich medium together with acLDL (acetylated low-density lipoprotein) for 7 days (macrophages) or 14 days (foam cells). In addition, the expression of the binding partners of Gremiin, namely BMP-2, -4 and -7 is shown. The expression of the BMP receptors IA and II was also investigated. All these "partners" of Gremiin could be detected in the different cell types of the arterial vessel wall.

Figure 1B shows the results of immunohistochemical staining demonstrating the expression of gremiin at the protein level: Immunofluorescent staining with anti-Gremlin human and secondary monocyte (B) and macrophage and foam cell (C) antibodies is shown. Monocytes, macrophages and foam cells were human stained simultaneously with anti-CD68.

Subsequently, the foam cell formation was studied starting from monocytes under the influence of Gremiin. For this purpose, monocytes were cultured together with platelets in serum-rich medium for 15 days. Murine gremlin protein was added in 3 different concentrations, namely 500, 1000 and 2000 ng / ml medium on day 1 of the culture. As control proteins, a protein similar to Gremiin, PRDC (protein related to DAN and cerberus, DAN and Cerberus related protein, also a BMP antagonist) was added at a concentration of 1000 ng / ml. In addition, the effect of the antagonists of Gremiin BMP-2, -4 and -7 in a concentration of 1000 ng / ml medium examined. Foam cells per visual field were scored on days 3, 6, 9, 12 and 15 from 5 representative visual fields, magnification 10x.

The results of these investigations are shown in Fig. 2: Fig. 2A shows the foam cell formation under the influence of 500, 1000 and 2000 ng Gremiin per ml medium: It was found that under the influence of Gremiin significantly less foam cells per visual field were present. Figure 2B shows the foam cell formation under the influence of Gremiin 1000 ng / ml and PRDC, BMP-2, -4 and -7, each at 1000 ng / ml in 5 independent foam cell cultures. It could be shown that only under the influence of Gremiin a significant decrease in foam cell counts occurred. PRDC and BMP-2 and -7 even led to a tendency to increase foam cell counts.

In further experiments, the supernatants from the foam cell cultures were examined in CytokinArray: For this purpose, monocytes were cultured together with platelets in serum-rich medium for 15 days. Murine skeletal protein was added to culture at a concentration of 2000 ng / ml and 5000 ng / ml medium on day 1. The control proteins used were again PRDC, BMP-2, -4 and -7, each at a concentration of 2000 ng / ml. The supernatants were collected on day 15 and frozen in CytokinArray at -20 ° C until analysis.

The results of these experiments are shown in Figure 3A. Under the influence of Gremiin, three of the cytokines tested, namely the Macrophage Migration Inhibitory Factor (MIF), CXCL1 and the Monocyte Chemoattractant Protein 1 (MCP-1 ) a reduced expression. The BMP proteins did not show this effect. PRDC also resulted in a decrease in MCP-1 expression; but it did not decrease the concentration of MIF and CXCL1. In this context, the concentrations of MIF and CXCL1 under the influence of gremiin were also examined in the supernatant of monocytes, macrophages and foam cells. For this purpose, first the supernatants from 5 independent experiments were obtained as described for under 3A and tested in anti-MIF and anti-CXCLl ELISA (enzyme-linked immunosorbent assay) (left panel in each case in Fig. 3B, 3C and 3D). Depending on the concentration of gremiin (2 g / ml and 5 pg / ml medium) there was an increasing decrease in the concentration of MIF (Figure 3B), MCP-1 (Figure 3C) and CXCL1 (Figure 3D). ,

To clarify whether Gremiin reduces the expression of MIF and CXCL1 also in monocytes and macrophages, monocytes were incubated with Gremiin, PRDC, BMP-2, -4 and -7 in the concentrations described above for 48 hours and their supernatants (n = 11), or they were cultured together with platelets for 7 days in serum-rich medium, so that macrophages formed from them. Again, the proteins were added at the above-described concentration on day 1 (n = 10). Gremiin resulted in a significant reduction in the concentration of MIF, measured in the anti-MIF ELISA, in both monocytes, macrophages and foam cells.

Further experiments to investigate the inhibition of MIF secretion by Gremiin showed (see FIG. 4) that after incubation of monocytes with Gremiin the MIF concentration is already reduced after less than 8 hours in comparison to controls (FIG. 4) , above). Even after stimulating the cells with LPS (lipopolysaccharide), MIF is already reduced in less than 8 hours by the addition of gremiin compared to controls (Figure 4, bottom). Thus, Gremiin inhibits the secretion of MIF from its intracellular stores.

In further studies, the apoptosis rate in macrophages and foam cells under the influence of Gremiin was examined. For this purpose, monocytes were co-administered with platelets in serum-rich medium for 15 days cultured. Murine gremlin protein was added at a concentration of 1000 ng / ml medium on day 12 of the culture. As control proteins, PRDC and BMP2, each at a concentration of 1000 hg / ml of medium on day 12 of culture, were added thereto. The control proteins used were PRDC and BMP2, each at a concentration of 1000 ng / ml medium. On day 14, a TUNEL assay was performed. As shown in Figure 5, there was a significant increase in apoptosis rate only under the influence of gremiin (p <0.05). PRDC and BMP2 did not cause significant apoptosis.

Further, the influence of human monocyte migration by Gremiin in the Boyden chamber was examined. For this purpose, monocytes were isolated from the peripheral blood of healthy adults and pipetted into the upper chamber of a Boyden chamber, pore size 5 m. In the lower chamber was medium with Gremiin in two different concentrations (2 and 5 g / ml) (n = 3). Depending on the Gremlin concentration, there was an increasing stimulation of monocyte migration. In the higher concentration (5 g / ml) Gremiin proved to be a stronger chemo-attractant than MCP-1. The combination of Gremiin and MCP-1 did not further increase monocyte migration. In contrast, the combination of gremiin and MIF reduced the migration of monocytes as compared to gremiin alone (see graph in Figure 6A above). The experiment was repeated 3 times, with MIF alone in the lower chamber serving as a control (10 and 50 ng / ml). The monocyte migration was the most inhibited by MIF. Gremiin proved to be a strong chemoattractant, as in the first 3 experiments, and a combination of gremiin and MIF took an intermediate position (Fig. 6A diagram below). Gremiin has thus opposite effects on monocyte migration as MIF.

In Figs. 6B and C, Gremiin is also shown to regulate the activity of MMP2: Monocytes were cultured in serum-rich medium along with acLDL for 14 days to form macrophages and foam cells. On day 1.5 and 10, Gremiin 1 g / ml was added to each. On day 14, the supernatants were collected and visualized using gelatin zymography. their matrix metalloproteinase activity (MMP2 and MMP9). Gremiin resulted in a significant reduction of MMP2, the activity of MMP9 was not significantly affected. The reduction in MMP2 activity is probably due to the reduction in MIF concentration, which is reduced by Gremiin. The positive control was ILIO (Interleukin 10), which stimulates the secretion of MIF.

Based on these findings, an embodiment of a fusion protein according to the invention was generated. For this purpose, the IgG ₂ Fc portion was fused via a spacer (linker peptide) and a thrombin interface with murine Gremiin (mGremlin). For expression in the cell supernatant, this construct was further fused with the IgK leader sequence, which is a secretion signal. The schematic overview shows that thus the C-terminus of IgG ₂ Fe is linked to the N-terminus of a spacer whose C-terminus is in turn connected to the N-temrinus of a thrombin interface whose C-terminus is in turn connected to the N-terminus. Terminus of Gremiin is linked. At the N-terminus of IgG ₂ Fe, the C-terminus of the secretion signal is fused. This structure is shown schematically in Flg. 7, wherein the amino acid sequence of this embodiment is shown in FIG. 7 below. The fusion protein is also referred to herein as mGremlin-Fc.

CHO cells (Chinese hamster ovary, CHO flp-In) were stably transfected with a vector containing the fusion gene (pCDNA5_FRT). To detect the proteins subsequently purified from cell culture supernatants by means of ProteinG agarose, a Western blot was prepared, using in each case 1 mg of the mGremlin-Fc, the IgK-Fc and the positive control (recombinant murine gremiin). The recombinant mGremlin and the mGremlin Fc were detected by means of an anti-gremlinl antibody (Abcam, ab45292), the IgK Fc by means of an anti-human IgG antibody (Jackson, ImmunoResearch, Pennsylvania, USA; No. 109-035-098). The results of this Western blot are shown in FIG. As can be seen from FIG. 8, the fusion protein could be successfully obtained from the culture supernatants. In further experiments, the binding ability of the fusion protein to BMP-4 was examined. For this purpose, a BMP-4 ELISA was carried out with increasing mGremlin-Fc concentrations. The results of this experiment are shown in Figure 9, after which a concentration-dependent binding of the protein Gremiin to BMP-4 could be demonstrated. The IgK Fc used for control has no binding activity, which could preclude any non-specific binding of the Fc portion.

Thus, in summary, these results demonstrate that Gremlin-Fc binds to BMP, that it reduces the formation / secretion of MIF and CXCL1 and MCP-1, and that it can inhibit the formation of foam cells that can control migration of monocytes, and inhibit the activity of matrix metalloproteinase 2, which is why the protein is a valuable agent for the treatment of inflammatory and immunomodulatory diseases.

Claims

claims

A protein having the sequence of gremiin, or variants or fragments thereof having the BMP binding function or the MIF binding function of gremiin, for use in the treatment of diseases associated with a reduction in MIF production and / or the CXCLl formation and / or the MCP-l formation are treatable.

2. Protein according to claim 1, for the treatment of atherosclerosis, ischemic heart disease, myocardial infarction, stroke, heart failure, myocarditis, inflammatory kadriomyopathy, inflammatory rheumatic and autoimmune diseases, for the regeneration of ischemic tissue after myocardial infarction, and in ischemia / Reperfusion after organ transplantation and organ infarction.

3. Protein according to one of claims 1 or 2, characterized in that it has an amino acid sequence which is selected from one of SEQ ID Nos. 2 or 3, or variants or fragments thereof, the BMP binding function or MIF binding function of Own Gremiin.

4. A fusion protein comprising a) a first polypeptide having an immunoglobulin Fc domain, and b) a second polypeptide selected from Gremiin or variants or fragments thereof having the BMP binding function of Gremiin.

5. A fusion protein according to claim 4, characterized in that it contains in the direction of the N-terminus to the C-terminus: a) a first polypeptide having an immunoglobulin Fc-domain, and

b) a second polypeptide selected from Gremiin or variants or fragments thereof having the BMP binding function of Gremiin.

6. fusion protein according to claim 4 or 5, characterized in that the Fe domain is a human immunoglobulin Fc domain.

7. Fusion protein according to one of claims 4 to 6, characterized in that the first polypeptide having the amino acid sequence with the SEQ ID NO. 1 has.

8. The fusion protein according to any one of claims 4 to 7, characterized in that the second polypeptide has an amino acid sequence selected from SEQ ID NO: 2 or 3, or variants or fragments thereof, which have the BMP binding function of Gremiin ,

9. fusion protein according to any one of claims 4 to 8, characterized in that first polypeptide is linked directly or via one or more linker peptides with the second polypeptide.

10. fusion protein according to claim 9, characterized in that the linker peptide contains the amino acid sequence SEQ ID NO. 4 from the attached sequence listing.

11. fusion protein according to any one of claims 9 or 10, characterized in that the linker peptide contains a thrombin interface.

12. Fusion protein according to claim 11, characterized in that the thrombin interface has the amino acid sequence SEQ ID No. 5 from the attached sequence listing.

13. Fusion protein according to one of claims 4 to 12, characterized in that it has the amino acid sequence with the SEQ ID Nos. 7, 8 or 9.

14. Nucleic acid molecule comprising a sequence selected from the group: a) the nucleic acid sequence coding for the fusion protein with SEQ ID NO.

7, 8 or 9, or a variant thereof, which codes for the same fusion protein according to the degeneracy of the genetic code; b) a nucleic acid sequence encoding a polypeptide having at least 70% sequence homology to the polypeptide encoded by SEQ ID NO: 7, 8 or 9, wherein the nucleic acid sequence encodes a polypeptide comprising from its N-terminus to its C-terminus a nucleic acid sequence encoding an immunoglobulin Fc domain, a nucleic acid sequence encoding a linker peptide, a nucleic acid sequence encoding a thrombin site, and a nucleic acid sequence encoding Gremiin or a functional variant thereof capable of binding to BMP; c) a nucleic acid which has, in the 5 'to 3' direction, a first section which codes for an immunoglobulin Fc domain or for a functional conservative variant thereof which is functional in such a way that it corresponds to a polypeptide coded by the nucleic acid. tid allows expression in a cell in a form capable of binding to BMP, a second stretch indicative of the amino acid sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly -Ser Gly-GlyGly-Gly-Ser, a third section encoding a thrombin site, and a fourth section encoding Gremiin.

15. A vector comprising a nucleic acid according to claim 14.

16. fusion protein encoded by a nucleic acid according to claim 14.

17. Cell, expressing a fusion protein according to any one of claims 4 to 13 or 16.

18. A pharmaceutical composition containing the protein according to any one of claims 1 to 3, or a fusion protein according to any one of claims 4 to 13 in a pharmaceutically effective amount, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.

19. A fusion protein according to any one of claims 4 to 13, or the fusion protein according to claim 16, or the pharmaceutical composition according to claim 18, for use as a medicament, in particular for the treatment of atherosclerosis, ischemic heart disease, myocardial infarction, stroke, heart failure, myocarditis of inflammatory kadriomyopathy, inflammatory rheumatic and autoimmune diseases, for the regeneration of ischemic tissue after mycardial infarction, and in ischemia / reperfusion after organ transplantation.