EP2552488A1 - Protéines de fusion et leurs utilisations - Google Patents

Protéines de fusion et leurs utilisations

Info

Publication number
EP2552488A1
EP2552488A1 EP11710763A EP11710763A EP2552488A1 EP 2552488 A1 EP2552488 A1 EP 2552488A1 EP 11710763 A EP11710763 A EP 11710763A EP 11710763 A EP11710763 A EP 11710763A EP 2552488 A1 EP2552488 A1 EP 2552488A1
Authority
EP
European Patent Office
Prior art keywords
fusion protein
gpvi
polypeptide
gly
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11710763A
Other languages
German (de)
English (en)
Inventor
Christoph Leder
Meinrad Gawaz
Melanie Ziegler
Konstantinos Stellos
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eberhard Karls Universitaet Tuebingen
Universitaetsklinikum Tuebingen
Original Assignee
Eberhard Karls Universitaet Tuebingen
Universitaetsklinikum Tuebingen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eberhard Karls Universitaet Tuebingen, Universitaetsklinikum Tuebingen filed Critical Eberhard Karls Universitaet Tuebingen
Publication of EP2552488A1 publication Critical patent/EP2552488A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • 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.
  • 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.
  • changes in the vessels can lead to infarcts or myocarditis.
  • the endothelium is the single layer vessel wall lining that separates the bloodstream from the thrombogenic structures of the subendothelium.
  • 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.
  • cardiovascular diseases such as, for example, angina or myocardial infarction
  • rapid reperfusion of ischemia-affected coronary arteries is of utmost importance to prevent injury to the myocardium.
  • irreversible damage to the myocytes arrests, halting functional metabolism in the myocardium, eventually leading to cell death from necrosis and apoptosis.
  • tissue, vessels or organs including myocardium
  • 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.
  • 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.
  • 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.
  • GPVI collagen-binding domain
  • 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.
  • 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.
  • SDF-1 Stromal Cell Derived Factor-1
  • GPVI glycoprotein VI
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • GPVI or SDF-1 proteins each having either the GPVI or the SDF-1 binding property, the identical or substantially equivalent.
  • 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.
  • 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 .
  • 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 .
  • 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.
  • 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.
  • 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.
  • MMP matrix metalloproteinase
  • SDF-1 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.
  • fusion protein 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.
  • 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.
  • Soluble GPVI shows only as a dimeric form, eg. In association with the immunoglobulin Fc domain, affinity for collagen.
  • 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.
  • 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 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.
  • 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.
  • cDNA libraries 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).
  • 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.
  • 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 ⁇ , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Fe domain is a human IgG Fc domain.
  • the dimerizing peptide is linked directly or via a second linker molecule / spacer to the second polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fusion proteins of the invention can be prepared using recombinant expression vectors known in the art.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • administration include parenteral, e.g. intravenous, intradermal, subcutaneous, transdermal, transmucosal administration.
  • parenteral e.g. intravenous, intradermal, subcutaneous, transdermal, transmucosal administration.
  • the fusion protein 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.
  • the pharmaceutical composition according to the invention is prepared for administration via a stent or balloon catheter.
  • 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.
  • 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.
  • G CSF granulocyte colony stimulating factor
  • dipeptidyl peptidase IV inhibitors are active ingredients selected from at least one of G CSF (granulocyte colony stimulating factor) or dipeptidyl peptidase IV inhibitors.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • a short (3 amino acid) linker positions 353 to 355
  • the fusion protein is still linked to the IgG2 Fc moiety (positions 356 to 578).
  • FIG. 1 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).
  • CHO cells from Chinese hamster ovary
  • Flp-In cells Invitrogen
  • the cells expressed the fusion protein in the supernatant from which it was purified.
  • a collagen GPVI ELISA was performed.
  • 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.
  • 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.
  • FIG 3 binding of SDF1-GPVI to CXCR4 was demonstrated by a FACS competition assay.
  • 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.
  • the fusion protein was examined for its chemotactic function.
  • 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.

Abstract

La présente invention concerne une protéine de fusion comprenant a) un premier polypeptide qui est sélectionnée parmi SDF-1 (facteur -1 dérivé de cellules stromales) ou des variantes résistantes à la peptidase/protéase ou des fragments de celles-ci qui ont la fonction de liaison CXCR4/CXCR7 de SDF-1; et b) un deuxième polypeptide qui est sélectionné parmi GPVI (glycoprotéine VI) ou le domaine extracellulaire de GPVI ou des fragments ou variantes du domaine extracellulaire de GPVI qui ont la fonction de liaison de collagène de GPVI, le premier polypeptide et le deuxième polypeptide étant reliés directement l'un à l'autre ou par l'intermédiaire d'une molécule de liaison. L'invention concerne également l'utilisation de la protéine de fusion pour traiter des maladies.
EP11710763A 2010-03-30 2011-03-23 Protéines de fusion et leurs utilisations Withdrawn EP2552488A1 (fr)

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DE102010013887A DE102010013887A1 (de) 2010-03-30 2010-03-30 Fusionsprotein und dessen Verwendungen
PCT/EP2011/054465 WO2011120859A1 (fr) 2010-03-30 2011-03-23 Protéines de fusion et leurs utilisations

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JP (1) JP2013523103A (fr)
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WO (1) WO2011120859A1 (fr)

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EP2377888A1 (fr) * 2010-04-07 2011-10-19 Corimmun GmbH Protéine de fusion
WO2016197491A1 (fr) * 2015-06-12 2016-12-15 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Échafaudage de tissu en trois dimensions avec élément d'attraction de cellules souches et utilisation correspondante
CN108368165A (zh) * 2015-06-24 2018-08-03 德克萨斯大学系统董事会 用于治疗颅内出血相关症状的方法和组合物
EP3424957A1 (fr) * 2017-07-03 2019-01-09 advanceCOR GmbH Protéine de fusion

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WO2002014505A1 (fr) * 2000-08-15 2002-02-21 Terumo Kabushiki Kaisha Polypeptide hybride de fixation du collagene
DE102007010306A1 (de) 2007-02-22 2008-08-28 Eberhard-Karls-Universität Tübingen Universitätsklinikum Bispezifisches Fusionsprotein mit therapeutischem und diagnostischem Potenzial

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WO2011120859A1 (fr) 2011-10-06
JP2013523103A (ja) 2013-06-17
DE102010013887A1 (de) 2011-10-06
US20130108580A1 (en) 2013-05-02

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