EP2552488A1

EP2552488A1 - Fusion protein and its uses

Info

Publication number: EP2552488A1
Application number: EP11710763A
Authority: EP
Inventors: Christoph Leder; Meinrad Gawaz; Melanie Ziegler; Konstantinos Stellos
Original assignee: Eberhard Karls Universitaet Tuebingen; Universitaetsklinikum Tuebingen
Current assignee: Eberhard Karls Universitaet Tuebingen; Universitaetsklinikum Tuebingen
Priority date: 2010-03-30
Filing date: 2011-03-23
Publication date: 2013-02-06
Also published as: WO2011120859A1; US20130108580A1; JP2013523103A; DE102010013887A1

Abstract

The present invention relates to a fusion protein comprising a) a first polypeptide selected from among SDF-1 (stromal cell derived factor-1) or peptidase/protease-resistant variants or fragments thereof which have the CXCR4-/CXCR7- binding function of SDF‑1; and b) a second polypeptide which is selected from among GPVI (glycoprotein VI), or the extracellular domain of GPVI, or fragments or variants of the extracellular domain of GPVI which contain the collagen binding function of GPVI where the first polypeptide and the second peptide are linked to one another directly or via a linker molecule. The invention furthermore relates to the use of the fusion protein for treating diseases.

Description

Fusion protein and its uses

The present invention relates to a fusion protein with therapeutic potential, in particular for the treatment or regeneration of lesions of vessels, organs or tissues, or for the improvement of hematopoiesis; The invention further relates to a nucleic acid molecule coding for this fusion protein, a pharmaceutical composition containing the fusion protein, and the use of the fusion protein or the pharmaceutical composition for the treatment of lesions of vessels or tissues, as well as of acute or chronic vascular diseases, or for the regeneration or renewal thereof , ie for angiogenesis, as well as for the improvement / support of hematopoiesis.

The pathological change of vessels and tissues of the human body can - in addition to physical injuries - different findings or diseases are based. These include, for example, injuries which may occur during implantation of stents or stent grafts, viral or bacterial infections. In the case of atherosclerosis, arterial degeneration also occurs as a change in the vessel walls, ie in particular growths and deposits, and various factors can contribute to their development. In the case of, for example, diseases of the heart muscle, changes in the vessels can lead to infarcts or myocarditis.

In general, damage in the vessel wall can lead to the removal of the integrity of the vessel wall and subsequent bleeding into surrounding tissue. To prevent this, platelets in combination with soluble plasma components form a hemostatic thrombus, which seals the damage and causes hemostasis. Once a lesion occurs on a vessel, it immediately initiates various cellular and biochemical mechanisms necessary for hemostasis. In arterial haemostasis, the endothelium also plays a central role by regulating the permeability to plasmalipoproteins, leukocyte adhesion and the secretion of pro- and antithrombotic factors as well as vasoactive substances.

The endothelium is the single layer vessel wall lining that separates the bloodstream from the thrombogenic structures of the subendothelium. In endothelial damage of the vascular wall and the now exposed thrombogenic subendothelial matrix occurs during hemostasis for the adhesion of resting, circulating in the blood platelets to now exposed collagen. This initial adhesion process is controlled by platelet membrane glycoprotein receptors, the integrins, resulting in a change in shape, platelet activation, and release of the ingredients from the storage granules. The thrombocytic glycoprotein VI (hereinafter also abbreviated to "GPVI") interacts directly with the exposed collagen and stabilizes the binding. GPVI, the most important collagen receptor, not only mediates tighter binding directly to collagen, but also mediates the activation of other receptors required for adhesion. After adhesion, the next step in hemostasis is aggregation, which leads to an accumulation of thrombocytes in the thrombus. Therefore plays in the activation Platelet GPVI as a collagen receptor on the platelet surface plays a crucial role and is also considered a risk factor for myocardial infarction. By the occurrence of such thrombi, the supply of blood to the tissue is no longer guaranteed, so that ischemic conditions of the tissue located distal to the thrombus can occur.

Thus, cardiovascular diseases, such as, for example, angina or myocardial infarction, currently still account for about one third of all worldwide deaths. In these diseases, rapid reperfusion of ischemia-affected coronary arteries is of utmost importance to prevent injury to the myocardium. As blood flow in a coronary vessel is reduced, irreversible damage to the myocytes arrests, halting functional metabolism in the myocardium, eventually leading to cell death from necrosis and apoptosis.

The regeneration of tissues, vessels or organs, including myocardium, depends strongly on the recruitment and accumulation of a small population of stem cells to the injured or diseased sites concerned. As a rule, injury to the tissues / organs / vessels is stimulated, as a result of which these stem cells increasingly circulate in the peripheral blood and adhere to the damaged areas. It is known that CD34 ⁺ stem cells from the bone marrow support the integrity of the vascular endothelium, as these can differentiate into endothelial cells after attachment to the affected site.

Targeted and controlled recruitment of these progenitor cells to affected sites would thus be a preferred tool to support natural re-endothelialization.

WO 2008/101700 discloses a bispecific fusion protein via which, on the one hand, targeted progenitor cells can be recruited to tissue / vessels by virtue of the collagen-binding domain (GPVI) being used to recruit the fusion protein. tein is bound to injured tissue / vascular sites, and can be recruited via the progenitor cell binding domain precursor cells.

However, there is still a great need for other, alternative products and substances, via which an improved recruitment of progenitor cells or CD34 ⁺ cells can take place at affected sites.

The object of the present invention is therefore to provide a new agent which can be used for the treatment of tissue and vascular diseases, in particular of the heart vessels, as well as for the regeneration of injured tissue or vessels.

According to the invention, this object is achieved by a fusion protein which comprises a) a first polypeptide selected from SDF-1 (Stromal Cell Derived Factor-1) or variants or fragments thereof which have the CXCR4 / CXCR7 binding function of Possess SDF-1; and b) a second polypeptide selected from GPVI (glycoprotein VI), or the extracellular domain of GPVI, or fragments or variants of the extracellular domain of GPVI that possess the collagen-binding function of GPVI.

The object underlying the invention is further solved by a pharmaceutical composition containing the fusion protein according to the invention in a pharmaceutically effective amount, optionally in combination with a pharmaceutically acceptable carrier, as well as by the use of the fusion protein according to the invention or the pharmaceutical containing it Composition as a medicament, in particular for the treatment of cardiovascular diseases, for endothelial regeneration in tissues, organs and vessels, and for the support of hematopoiesis and angiogenesis.

The fusion protein according to the invention can via the second polypeptide, namely the binding to collagen GPVI - or via the extracellular Domain of GPVI, or fragments or variants of the extracellular domain of GPVI that have the collagen-binding function of GPVI - bind to collagen, and the first polypeptide, namely SDF-1 (stromal cell derived factor-1) or variants or fragments thereof which have the CXCR4 / CXCR-7 binding function of SDF-1, bind CD34 ⁺ cells that have the receptors of SDF-1, CXCR4 or CXCR7, and thus recruite to sites where collagen is exposed, ie in particular injured vessels, organs or tissues. The progenitor cells recruited from the fusion protein according to the invention to lesions can subsequently differentiate into endothelial cells and serve for the regeneration - and finally for the treatment of a disease of the tissue, organ or vessel.

In the present context, a "fusion protein" is understood as meaning a hybrid protein or an artificial protein, which can be synthesized in vitro or in vivo by molecular biological or chemical methods known in the art by combining or linking two (or more) (poly) peptides , otherwise or of course not connected or linked together and otherwise not occur naturally, can be produced. 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 term "fragment" or a "variant" which has the binding function of the respective polypeptide is understood here to mean an amino acid sequence which differs from the wild-type sequence or the sequence given here 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 can also Amino acid deletions and / or insertions include. For example, guidance for determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found, for example, using computer programs known in the art. The presently encompassed protein or polypeptide variants or fragments thereof include GPVI or SDF-1 proteins, each having either the GPVI or the SDF-1 binding property, the identical or substantially equivalent. For example, whether a modified GPVI or SDF-1 polypeptide possesses the binding properties of the unmodified GPVI or SDF-1 polypeptides can be assayed in in v / fro assays, as described further in the present application. 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 sequence identity of two amino acid or nucleic acid sequences, it is possible to proceed according to methods known in the art, for example by means of an alignment and calculation by means of a mathematical algorithm.

By "linker" or "spacer" or "linker molecule", which are used interchangeably herein, in the present invention, an amino acid or a nucleic acid sequence encoding it is understood to be up to 100 amino acids / bases, which serves to link two functional polypeptides, and which optionally generates a distance between the two functional polypeptides without possessing bioactivity or binding property to other molecules. This is therefore a "neutral" sequence, which can fulfill or fulfill no other than the functions just described.

In particular, it is provided in a preferred embodiment, when in the fusion protein according to the invention, a peptidase / protease, in particular a dipeptidyl peptidase IV resistant variant of SDF-1 is used as the first polypeptide, in particular a matrix metalloproteinase resistant variant. Among other things, SDF-1 can be cleaved by the matrix metalloproteinase (MMP) -2, thereby losing its chemotactic bioactivity. This can be prevented by using a modified SDF-1 variant which is resistant to MMP-2 but which retains its chemotactic bioactivity. An example of this SDF-1 variant is, for example, by Segers et al., ("Local Delivery of Protease-Resistant Stromal Cell Derived Factor-1 for Stern Cell Recruitment After Myocardial Infarction", Circulation, 2007, 116: 1683-1692 ); this is referred to as S-SDF-1, and this is hereby incorporated by reference.

The inventors of the present application were able to show in their own experiments that the fusion proteins according to the invention could bind to soluble collagen and to the CXCR4 receptor of certain cells. Furthermore, it could be shown that the fusion protein was able to stimulate the chemotaxis of hematopoietic stem cells. This shows that CXCR4 ⁺ precursor cells can be recruited by the fusion protein and that the SDF-1 portion of the fusion protein is still functional. With the fusion proteins according to the invention it is then possible, in a simple and targeted manner, the re-endothelialization and the restoration of injured vessels or any tissue that releases or exposes collagen by injury or other influences on its surface, through the colonization with To treat stem cells and their maturation.

Furthermore, it is also possible to improve hematopoiesis with the fusion protein according to the invention, which is particularly required in bone marrow ablation or transplantation. The fusion proteins according to the invention can bind to collagen binding sites for GPVI, which are accessible in the mentioned interventions after chemotherapy or radiotherapy, accumulate in the bone marrow and promote and support the repopulation of the bone marrow and blood cell formation at these sites by recruiting progenitor cells of stem cells. Endothelial progenitor cells are a circulating bone marrow-derived cell population of large non-leukocyte cells involved in vascular repair and hemostasis.

The receptor GPVI is the most important receptor of the platelets for collagen. GPVI allows the aggregation, secretion, shape change and activation of platelets. Human GPVI contains a 20 amino acid signal sequence, an extracellular domain of 247 amino acids, as well as a 21 amino acid transmembrane domain and a 51 amino acid long cytoplasmic tail.

Since binding to collagen is mediated via the extracellular domain, in one embodiment of the fusion protein of the invention, it is preferred that the first polypeptide has the extracellular portion of GPVI, or fragments or variant of the extracellular domain of GPVI, that has the collagen binding function of GPVI linked to a dimerizing peptide.

[0024] It is advantageous here that it is possible, for example, to resort to already soluble GPVI previously described in the prior art (see Massberg et al., "Soluble glycoprotein VI dimer inhibits platelet adhesion and aggre- gation to the injured vessel Wall in vivo ", FASEB J. 2004; 18: 397-399, which reference is made to this publication for the preparation of soluble human GPVI.

Soluble GPVI shows only as a dimeric form, eg. In association with the immunoglobulin Fc domain, affinity for collagen. To generate this soluble GPVI, the extracellular portion of the human GPVI can be cloned and linked to the human immunoglobulin Fc domain. This GPVI-Fc protein (also referred to below as soluble GPVI-Fc) can be expressed, for example, with the aid of adenoviruses via a human HeLa cell line. With such a soluble GPVI-Fc was able to detect collagen adhesion both in vitro and in vivo.

It is understood by one skilled in the art that in order to fulfill the function of the invention, namely with respect to the GPVI binding to collagen, the fusion protein does not necessarily have to use the complete or identical amino acid sequence of the soluble GPVI. Rather, the function of the fusion protein according to the invention is also fulfilled if the second polypeptide has a segment or a sequence variant of the soluble GPVI which, however, still exerts the collagen-binding function of GPVI 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 the first polypeptide is a variant of soluble GPVI in which one or more amino acids of one of 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 soluble GPVI.

"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 responsible for the dimerization function. Advantageously, recourse may be had to commercially available human or mouse Fc DNA, which can either be isolated from commercially available cDNA libraries by PCR, or which is already cloned into plasmids, which in turn are commercially available can be purchased (for example, available from Invitrogen, San Diego, USA).

It is understood that a fragment or a variant of the Fc domain can be used without affecting the function of the second polypeptide according to the invention, as long as the fragment or the variant still has the possibly attenuated dimerization function of an antibody; see. above statements on fragments or variants of GPVI that apply equally to the fragment or variant of Fe.

Incidentally, any other molecule with dimerization function is also suitable to be included in the present fusion protein, as long as thereby the dimerization of GPVI is ensured. It will be clear to those skilled in the art that the corresponding sequence of another dimerization molecule can be incorporated into the fusion protein instead of the Fc portion.

The dimerization molecule is designed in terms of its amino acid sequence to have a portion of a protein involved in mediating dimerization of two separate proteins or protein subunits. This measure too is easy for the person skilled in the art since the fine structures, including the amino acid sequences of peptidic dimer complexes, are described in detail for a large number of proteins in the prior art. Known dimer-forming proteins known in their sequence and structure include G proteins, histones, interferon γ, interleukin-2 receptor, Hsp90, tyrosine kinases, IgG molecules, etc. The respective dimerization-mediating domains of said proteins may be used to produce the present invention Fusion protein are taken directly. However, it may well be desirable to modify these domains by targeted mutagenesis or else by C- and / or N-terminal addition of individual amino acids, so that, for example, the immunological effect of the fusion protein is reduced, the better preparation. the fusion protein is allowed, but the dimerization function is largely retained.

In one embodiment, it is preferred if a variant of the Fc domain or a synthetic Fc fragment is used, which is mutated in the complement and Fc receptor binding region such that activation of the immune system is largely reduced and possibly even absent , Thus, for example, an Fc fragment can be used in which by targeted mutagenesis at position 331 a proline exchanged for a serine and at the amino acid positions 234 to 237 the tetrapeptide Leu-Leu-Gly-Gly for Ala-Ala-Ala-Ala becomes.

In a first embodiment, it is preferred if the first polypeptide has an amino acid sequence having SEQ ID NO: 1, 2 or 3 from the attached sequence listing.

The amino acid sequence designated by SEQ ID NO: 1 shows the sequence of human SDF-1, the amino acid sequence designated by SEQ ID NO: 2 shows the isoform SDF-1 alpha (without leader sequence) corresponding to SEQ ID No. 3 designated amino acid sequence is the isoform SDF-1 beta (without leader sequence). SDF-1 is post-translationally modified, in particular the leader sequence, shown in SEQ ID NO: 8, cleaved off. SDF-1 is an endogenous chemokine from the group of CXC motif chemokines, and is also referred to as CXCL12. SDF-1 binds to the chemokine receptors CXCR4 and CXCR7, which belong to the family of G protein-coupled receptors and are activated by the binding of SDF-1.

In a further embodiment it is preferred if the second polypeptide has an amino acid sequence with SEQ ID NO: 4 from the attached sequence listing. The amino acid sequence SEQ ID NO. Figure 4 represents the extracellular domain of the human GPVI, including two additional amino acids of the Trnas membrand domain. It is understood that variants or fragments thereof which possess the collagen-binding function of GOVI are also suitable for the purposes of the present invention. The person skilled in the art can, for example, resort to the variants already known in the prior art (as can be found, for example, in the UniProt / SwissProt Dantenbanken (www .uniprot.org)), or by way of obvious obvious experiments such fragments / variants generate, for example. Aminoäsurenuren exchanges, deletions, -isertionen.

As mentioned above, it is particularly preferred if the second polypeptide is the extracellular domain of GPVI, or a fragment or variant of the extracellular domain of GPVI that has the collagen-binding function of GPVI, and that the second Polypeptide is linked to a dimerizing polypeptide, in particular with an Fc-domain of an immunoglobulin or a fragment or a variant thereof, which has the dimerization function of the Fc-domain.

In this case, in a preferred embodiment, the Fe domain is a human IgG Fc domain.

In further embodiments, it is preferred if the dimerizing peptide is linked directly or via a second linker molecule / spacer to the second polypeptide. In one embodiment, it is preferred if the second linker molecule has the sequence glycine-glycine-arginine. 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 dimerizing peptide to the second polypeptide. According to a further embodiment, the linker molecule, via which the first polypeptide is linked to the second polypeptide in a preferred embodiment, has the sequence SEQ ID No. 5 from the attached sequence listing. It is understood that other linker molecules can be used for linking the two polypeptides, in particular those which lead to no or only a slight change in the biological activity of the corresponding fusion protein in comparison to the indicated linker molecule, so that such an amino acid exchange is the fusion protein according to the invention largely untouched in its function.

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. 6 or 7. The two sequences differ in that the sequence designated by SEQ ID No. 6 has a sequence for the secretion signal, whereas the sequence with SEQ ID No. 7 does not include this sequence.

The invention further relates to a nucleic acid molecule comprising a sequence selected from the group: a) the nucleic acid sequence coding for the fusion protein having the SEQ ID NO.

6 or 7, or a variant thereof, which codes for the same polypeptide 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: 6, said nucleic acid sequence encoding a polypeptide comprising from its N-terminus to its C-terminus SDF-1, a nucleic acid sequence encoding a first linker molecule, the extracellular domain of GPVI, or a variant thereof capable of binding to collagen, a nucleic acid sequence encoding a second linker molecule, and a dimerizing protein; lypeptide-encoding nucleic acid sequence which is operable to allow a protein encoded by the nucleic acid to be expressed in a cell in a form capable of binding to collagen and / or CXCR4 or CXCR7; c) a nucleic acid coding for a polypeptide which has a first section in the 5'-3 'direction which encodes SDF-1 or peptidase / protease-resistant variants or fragments thereof which contain the CXCR4 / CXCR7- Having a binding function of SDF-1, a second section encoding the amino acid sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-GlyGly-Gly-Ser, a third section coding for an extracellular domain of GPVI or a fragment or variant of the extracellular domain of GPVI having the collagen binding function of GPVI, a portion coding for a linker molecule having the sequence Gly-Gly-Arg, and a a fourth portion encoding a Fc domain or a functional conservative variant thereof, which is operable to allow a protein encoded by the nucleic acid to be expressed in a cell in a form capable of: at To bind collagen and / or CXCR4 or CXCR7.

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 which an array of one or more genetic elements having a regulatory role in gene expression, for example promoters, operators or enhancers, operably linked to a DNA sequence encoding the fusion protein of the invention which transcribes into mRNA and translates into the protein and a suitable transcriptional and translational start 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).

According to a further embodiment, the invention also relates to a pharmaceutical composition containing a fusion protein according to 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 with optionally further additives are well known in the art and are, for example, in the essay of Kibbe A., Handbook of Pharmaceutical Excipients, Third Edition, American Pharmaceutical Association and Pharmaceutical Press 2000. Additives in accordance with the invention include any compound or composition which is advantageous for therapeutic use of the composition, including 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 the GPVI domain in the area of the endothelial lesions, resulting in an SDF-I gradient and recruiting stem cells become. This provides a highly effective tool for the treatment of diseases caused by the lesion of vessels, organs or tissues which, as a result, exposes thrombogenic subendothelium.

In one embodiment, it is preferred if the pharmaceutical composition according to the invention is prepared for administration via a stent or balloon catheter.

Alternatively, it is preferred if the composition of the invention or the fusion protein is co-incubated with a stem cell solution - and thus the stem cells can bind to the fusion protein - and the fusion protein-precursor cell conjugates thus obtained are administered.

In particular, the present invention also relates to a pharmaceutical composition containing the fusion protein according to the invention in combination with an active ingredient which is selected from at least one of G CSF (granulocyte colony stimulating factor) or dipeptidyl peptidase IV inhibitors.

It is known that dipeptidyl peptidases IV inactivate SDF-1, so that a combination preparation of fusion protein and dipeptidyl peptidase IV inhibitors extends the half-life of SDF-1. On the other hand, it is known that G-CSF causes the mobilization of stem cells. Therefore, with a combination of the fusion protein and G-CSF, the binding rate of progenitor cells to the fusion protein can be increased.

The invention further relates to the use of the fusion protein or the pharmaceutical composition according to the invention for the treatment of diseases or for regeneration, in particular of vessels or tissues, or for the improvement of hematopoiesis and angiogenesis.

As already stated above, can be treated with the inventive fusion protein or a pharmaceutical composition containing this tissue, vessels or organs in which, for example, due to injury or disease subendothelium is exposed, so that on the one hand, the fusion protein GPVI can share in the thus exposed collagen, and on the other hand can be recruited via the SDF-1 portion CXCR4 + cells, so in particular precursor cells of stem cells to the injured sites. There, the precursor cells differentiate into endothelial cells and thus contribute to the re-endothelialization or to the healing of the diseased tissue / organ / vessel.

In particular, it can treat diseases selected from cardiovascular diseases, arteriosclerosis, myocarditis, myocardial infarction; Furthermore, the fusion protein according to the invention can be used for the regeneration of the myocardium, the blood-brain barrier in chronic progressive multiple sclerosis, for the treatment of fibrotic liver segments, vascular epithelium, in particular after stent implantation or in endothelial infections, after bone marrow deposition. tions, or tissue and vascular wounds in diabetes. Thus, inter alia, successful lesions of vessels can be treated, such as coronary vessels, brain-supplying vessels, extremity-supplying vessels, connective tissue, bone, as well as any vessel or tissue that has collagen.

It is understood that the features mentioned above and the features to be specified further below are possible not only in the particular combination specified, but also in other variations or in isolation, without departing from the scope of the present invention.

The invention is explained in more detail in the following example and in the figures.

Fig. 1 shows the schematic structure of an embodiment of the SDF-1-GPVI fusion protein (A) according to the invention; and protein expression of this embodiment (B), wherein the three domains of the embodiment of the fusion protein could be detected by immunoblot analysis with appropriate antibodies (anti-SDF-1, anti-GPVI, anti-IgG); the amino acid sequence of the embodiment shown schematically in Figure 1A is shown in (C), including a secretion signal sequence;

Fig. 2 shows the detection of collagen binding by ELISA (enzyme-linked immunosorbent assay) (A); which binding was competitive by incubation with soluble collagen (B);

Fig. 3 shows the binding of the embodiment of the fusion protein to CXCR4 on CD 14+ monocytes, represented by FACS (flow cytometry) competition analyzes; and 4 shows the concentration-dependent excitation of the chemotaxis of human hematopoietic stem cells by the fusion protein according to the invention in a Transwell system.

FIG. 1 shows a diagram of an embodiment of the fusion protein according to the invention in FIG. 1A, wherein the human SDF-1 successively from the N-terminus to the C-terminus is followed by a linker molecule via which SDF-1 is linked to GPVI. The linker molecule in this embodiment consists of the amino acid sequence (glycine ₄ serine) ₃ . The GPVI portion is linked to the human IgG2 Fc portion which causes dimerization.

In Fig. 1B immunoblots are shown, via which the expression of the individual components in the fusion protein, as shown schematically in Fig. 1A, was detected. On the left, by means of an anti-SDF-1 antibody (monoclonal anti-human / mouse CXCL12 / SDFlalpha antibody; R & D Systems, Minneapolis, USA), the SDF-1 portion was detected in the fusion protein, centered by an anti-GPVI antibody Anti-GPVI portion and right by means of an anti-IgG antibody, the IgG2 Fc portion in the fusion protein (first lane). It can be seen that the fusion protein has a size of about 85 kDa. Positive controls used for the detection of SDF-1 were the unfused polypeptide hSDF-1 (third lane), or the GPVI-FcIgG2 construct (for GPVI, third lane) or FcIgG2 alone (for FcIgG2, third lane).

In Fig. IC, the sequence of the fusion protein is shown above, which also has at its 5 'end still a 20 amino acid secretion signal sequence (IgK leader sequence) (SEQ ID No. 6); SEQ ID NO. Figure 7, shown in Figure 1C below, shows the fusion protein without this secretion signal sequence. This secretion signal sequence is responsible for the export of the fusion protein into the cell culture supernatant. Following this secretory signal sequence, amino acid position 21 to 88 is followed by an SDF-1 sequence (without leader) that is 68 amino acids long. The leader sequence of SDF-1 (MNAKVVVVLV LVLTALCLSDG; SEQ ID No. 8) is, as mentioned above, not included. Thereupon (positions 89 to 103 in SEQ ID No. 6) is followed by a 15 amino acid linker / linker sequence via which the extracellular GPVI domain (position 104 to 352) is linked to the fusion protein. This is followed in turn by a short (3 amino acid) linker (positions 353 to 355), via which the fusion protein is still linked to the IgG2 Fc moiety (positions 356 to 578).

The embodiment shown in FIG. 1 was generated by means of PCR and primers suitable in each case for the individual sections. The synthesized gene was then cloned into the vector pCDNA5 / FRT (Invitrogen). Subsequently, CHO (cells from Chinese hamster ovary) Flp-In cells (Invitrogen) were stably transfected with the construct. The cells expressed the fusion protein in the supernatant from which it was purified.

To obtain the data shown in Fig. 2, a collagen GPVI ELISA was performed. For this purpose, a 96-well plate was coated with 10pg / ml collagen, blocked with blocking solution and then incubated with the fusion protein or the corresponding control proteins. Subsequently, it was detected with the peroxidase-conjugated anti-human IgG antibody. In the following, the values of the binding curves were determined by measuring the wavelength at 450 nm. It can be seen in Fig. 2A, which binds both the fusion protein SDF1-GPVI and the control construct GPVI-FcIgG2 concentration-dependent on collagen, while FcIgG2 has no binding.

In Fig. 2B it could be shown that by pre-incubation of the fusion protein with soluble collagen as a competitive inhibitor binding was almost completely blocked.

In Figure 3, binding of SDF1-GPVI to CXCR4 was demonstrated by a FACS competition assay. For this purpose, monocytes were isolated because monocytes express CXCR4 on their surface. These monocytes were labeled with the fusiform incubated onprotein or the corresponding control proteins and subsequently stained with the aCXCR4-PE labeled antibody (BD Biosciences, catalog number 555974, USA). The binding of the fusion protein SDF1-GPVI to CXCR4 competes for the antibody and thus the aCXCR4-PE positive stained cells decreased in the subsequent FACS analysis. This increases the number of cells analyzed in the lower right quadrant. It was shown that the fusion protein SDF1-GPVI binds to CXCR4.

In Fig. 4, the fusion protein was examined for its chemotactic function. For this purpose, the fusion protein SDF1-GPVI in different concentrations (2 pg / ml, 10 pg / ml, 20 pg / ml), hSDFl as a positive control and medium as a negative control was placed in the lower chamber of a Transwell plate. CD34 ⁺ hematopoietic stem cells were isolated and added to each 150,000 cells in the upper chamber. After incubation for 6 h at 37 ° C in the incubator, the upper chamber was discarded. The cells migrated into the lower chamber were photographed and then the number of cells in the lower chamber was determined. For this purpose, the cells from the lower chamber were each counted for 1 min using the FACS. This experiment has shown that the fusion protein SDF1-GPVI is chemotactically effective.

Claims

claims

1. A fusion protein comprising a) a first polypeptide selected from SDF-1 (Stromal Cell Derived Factor-1) or variants or fragments thereof having the CXCR4 / CXCR7 binding function of SDF-1; and b) a second polypeptide selected from GPVI (glycoprotein VI), or the extracellular domain of GPVI, or fragments or variants of the extracellular domain of GPVI that possess the collagen binding function of GPVI, wherein the first polypeptide and the second Peptide are linked together directly or via a first linker molecule.

2. Fusion protein according to claim 1, characterized in that the first polypeptide has an amino acid sequence which is selected from the SEQ ID Nos. 1, 2 or 3, or variants or fragments thereof, the CXCR4 / CXCR7 binding function of SDF -1 own.

3. fusion protein according to claim 1 or 2, characterized in that a peptidase / protease resistant variant of SDF-1 is used, in particular a matrix metalloproteinase and / or dipeptidyl peptidase IV resistant variant.

4. fusion protein according to any one of the preceding claims, characterized in that the second polypeptide has an amino acid sequence which is selected from SEQ ID NO. 4 or 5.

5. The fusion protein according to any one of the preceding claims, characterized in that the second polypeptide is the extracellular domain of GPVI, or a fragment or variant of the extracellular domain of GPVI, which has the collagen-binding function of GPVI, and that the second polypeptide with linked to a dimerizing polypeptide.

6. The fusion protein according to claim 5, wherein the dimerizing polypeptide has an Fc domain of an immunoglobulin or a fragment or a variant thereof which has the dimerization function of the Fc domain.

7. fusion protein according to claim 6, characterized in that the Fe domain is a human IgG Fc domain.

8. fusion protein according to any one of claims 5 to 7, characterized in that dimerizing peptide is linked directly or via a second linker molecule with the second polypeptide.

9. fusion protein according to any one of the preceding claims, characterized in that the first linker molecule has the sequence SEQ ID NO. 5 from the attached sequence listing.

10. fusion protein according to any one of the preceding claims, characterized in that it has the amino acid sequence with the SEQ ID NO. 6 or 7.

11. Homodimer comprising the fusion protein according to any one of the preceding claims.

12. A nucleic acid molecule comprising a sequence selected from the group: the nucleic acid sequence encoding the fusion protein of SEQ ID NO: 6 or 7 or a variant thereof encoding the same fusion protein according to the degeneracy of the genetic code; a nucleic acid sequence encoding a polypeptide having at least 70% sequence homology to the polypeptide encoded by SEQ ID NO: 6, wherein the nucleic acid sequence encodes a polypeptide comprising from its N-terminus to its C-terminus SDF-1, a polypeptide a nucleic acid sequence encoding a first linker molecule, the extracellular domain of GPVI or a variant thereof capable of binding to collagen, a nucleic acid sequence encoding a second linker molecule, and a nucleic acid sequence encoding a dimerizing polypeptide that is functional allowing a protein encoded by the nucleic acid to be expressed in a cell in a form capable of binding to collagen and / or CXCR4 or CXCR7; a nucleic acid encoding a polypeptide having in the 5'-3 'direction a first portion encoding SDF-1 or peptidase / protease resistant variants or fragments thereof which express the CXCR4 / CXCR7 binding function of SDF-1, a second section encoding the amino acid sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-GlyGly-Gly-Ser, a third section dedicated to extracellular Domain of GPVI or a fragment or variant of the extracellular domain of GPVI that possesses the collagen-binding function of GPVI, encodes a portion encoding a linker molecule having the sequence Gly-Gly-Arg, and a fourth portion which encodes a Fc domain or a functional conservative variant thereof, which is operable to allow a protein encoded by the nucleic acid to be expressed in a cell in a form capable of contacting Koll agen and CXCR4 or CXCR7.

13. A vector comprising a nucleic acid according to claim 12.

14. fusion protein encoded by a nucleic acid according to claim 12.

A cell expressing a fusion protein according to any one of claims 1 to 11 or 14.

16. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 11 in a pharmaceutically effective amount, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.

17. A pharmaceutical composition according to claim 16, characterized in that it is in combination with an active ingredient selected from at least one of the following: G-CSF (granulocyte colony stimulating factor) or dipeptidyl peptidase IV inhibitors.

18. Use of the fusion protein according to any one of claims 1 to 11, or the fusion protein according to claim 14, or the pharmaceutical composition according to claim 16 or 17, as a medicament, in particular for the treatment of diseases or for regeneration, in particular of vessels or tissues, or Improvement of hematopoiesis and angiogenesis.

19. Use according to claim 18, characterized in that the diseases are selected from cardiovascular diseases, arteriosclerosis, myocarditis, myocardial infarction, dilated cardiomyopathy.

20. Use according to claim 19, characterized in that it is used for the regeneration of the myocardium, the blood-brain barrier in chronic progressive multiple sclerosis, fibrotic liver sections, vascular endothelium, in particular after stent implantations or in endothelial infections, according to Kno lesions, or tissue and vascular wounds in diabetes.