EP1315760A2 - Endoglin-specific polypeptide, production and use thereof - Google Patents

Abstract

The invention relates to a polypeptide which specifically binds to the extracellular domain of the human endoglin (CD105) protein, and the production and use thereof.

Description

 Endoglin specific polypeptide, its production and use

The present invention relates to a polypeptide which binds specifically to the extracellular domain of the human endoglin (CD 105) protein, and to its production and use.

Vascular target control, i.e. The selective recognition of cells or structures of the vascular bed represents a relatively new concept in medicine. This is intended to ensure that certain components that can be used diagnostically or therapeutically are specifically transported into the vascular bed. This approach is used among others in tumor therapy (Thorpe & Burrows, 1995, Breast Cancer Res. Treat. 36, 237-251). Here, e.g. by linking to a cytotoxic component that specifically attacks and eliminates the tumor vascular bed. This leads to an interruption in the supply of oxygen to the tumor tissue (hypoxia) and nutrients. The result is necrotization of the tumor. This approach is also used in gene therapy, e.g. for the targeted transduction of endothelial cells with gene therapy vectors (e.g. viruses, liposomes, DNA-protein complexes) (Wickham et al., 1997, J.Virol. 71, 8221-8229).

A requirement for vascular targeting is ligands that recognize specific structures in the vascular bed. Examples include peptides or proteins that bind to certain receptors or other surface molecules on the endothelial cells. Such receptors are, for example, the VEGF receptors or the α _v integrins (Burrows & Thorpe, 1994, Pharmac. Ther. 64, 155-174). Antibody fragments that recognize specific structures in the vascular bed can also be used. For example, endoglin (CD105), which is a member of the TGF-ß family, is significantly overexpressed by cells of the proliferating tumor endothelium (Miller et al., 1998, Int. J. Cancer 81, 568-572). Antibodies directed against endoglin have been described in the literature. The monoclonal antibody (MAb) SN6 was obtained by immunizing mice with cell membranes of human leukemia cells (Haruta & Seon, 1986, PNAS 83: 7898-7902). The MAb 44G4 was obtained by immunizing mice with human pre-B leukemia cells (Gougos & Letarte, 1988, J. Immunol. 141: 1925-1933). The MAb TEC4 and TECH were obtained by immunizing mice with human umbilical cord endothelial cells (HUVEC) (WO 96/01653). The MAb K4-2C10, D4-2G10, Y4-2F1 and P3-2G8 were obtained by immunizing mice with purified human endoglin (WO 97/45450). All previously known antibodies directed against human endoglin are thus derived from mice and, as a rule, lead to the formation of human anti-mouse antibodies (HAMA) in therapeutic applications in humans, which lead to the neutralization of the therapeutic antibodies. The use of antibodies from the mouse or other organisms for therapeutic purposes is therefore very limited.

An object of the present invention was therefore to provide a polypeptide which binds specifically to CD 105 and which does not lead to the formation of neutralizing HAMAs.

Another object of the present invention was to provide a polypeptide which is suitable for recruiting, for example, cytotoxic substances, liposomes or viruses on tumor endothelium.

Surprisingly, a polypeptide has now been isolated in the present invention which significantly improves the infection of human endothelial cells with an adenovirus.

The invention accordingly relates to a polypeptide which binds specifically to the extracellular region of human endoglin protein (CD 105), the Polypeptide contains one or more sequences according to SEQ ID No. 1. The extracellular region of the human endoglin protein comprises amino acids 1-559. Specific binding to human endoglin in the sense of the invention exists, for example, when the polypeptide is able to precipitate endoglin from a cell suspension or to detect endoglin in an ELISA. The specific binding of a polypeptide according to the invention is preferably determined by inhibiting (= competition) the binding of scFv C4, ie the binding is represented indirectly by inhibiting a protein with the same binding property. A polypeptide according to the invention then binds specifically to endoglin when a 1000- fold molar excess of the polypeptide compared to scFv C4 leads to essentially complete inhibition of the binding of scFv to endoglin. Preferably, essentially complete inhibition of binding occurs at a 100-fold molar excess, more preferably at a 50-fold molar excess. In a typical experiment to demonstrate the specificity of the binding of a polypeptide according to the invention, scFv C4 is used in a concentration of 1 μmol and the polypeptide is added in various concentrations in the range from 1 μmol to 1 mmol. Alternatively, one can use labeled polypeptides

In a further embodiment, the polypeptide additionally contains one or more sequences according to SEQ ID No. 2.

In one embodiment of the polypeptide according to the invention, the N and C terminals are each attached to the sequence according to SEQ ID No. 1 and the sequences according to SEQ ID. Nos. 1 and 2 add one to three cysteine residues, preferably one cysteine residue. This residue serves to stabilize the polypeptide. In a preferred embodiment, a peptide linker is inserted between every two sequences. The peptide linker is preferably between about 12 and about 25 amino acids in length. In a preferred embodiment, the polypeptide which contains one or more sequences as shown in SEQ ID No. 1 contains one or more amino acid domains of a human antibody, these amino acid domains from the framework region-1 (FR-1), FR-2, FR-3, FR-4, the complementarity-determining region-1 (CDR-1) and / or CDR-2 of the antibody, preferably from FR-1 to FR-4, CDR-1 and / or CDR-2 of the variable heavy chain (V _H ) are selected. The framework regions of the variable light (V _L ) or heavy chains have only a low sequence variability and they have a framework function in the antibody by which the spatial structure is determined. The complementarity-determining regions have a very high sequence variability within the variable domain of the light or heavy chain regions. The structure of the CDRs (CDR-1, CDR-2 and CDR-3) determines the binding specificity of the antibody. In a preferred embodiment, the polypeptide of the present invention contains, in addition to at least one sequence as shown in SEQ ID No. 1, FR-1 to FR-4, CDR-1 and CDR-2. These regions are particularly preferably selected from the V _H.

In a further embodiment, the polypeptide which contains one or more sequences according to SEQ ID No. 1 and SEQ ID No. 2 contains one or more amino acid domains of a human antibody, these amino acid domains from the framework region 1 (FR-1), FR-2, FR-3, FR-4, the complementarity-determining region-1 (CDR-1) and / or CDR-2 of the antibody are selected. Preferably, the amino acid sequence according to SEQ ID No. 1 with the FR-1 to FR-4, CDR-1 and / or CDR-2 of the V _H and the amino acid sequence according to SEQ ID No. 2 with the FR-1 to FR-4 , CDR-1 and / or CDR-2 of the V _L , the SEQ ID No. 1 and SEQ ID No. 2 taking the position of the CDR-3 in the VH and V _L , respectively.

The term “human” in the sense of the present invention relates to antibodies whose amino acid sequence has a high degree of homology to the variable regions of the human heavy (V _H ) and / or light chains (VL) and which are therefore not immunogenic in humans or only to a small extent. A high homology means that at least 80%, preferably 90%, particularly preferably 95%, in particular 98% of the amino acid residues are homologous. The degree of homology mentioned preferably relates to FR-1, FR-2, FR-3 and / or FR-4 while CDR-1 and CDR-2 domains do not have high homology. Because of the low immunogenicity, the polypeptide according to the invention, especially for therapeutic applications, has clear advantages over the murine antibodies described so far, since no neutralizing antibodies are formed.

The homology can be determined, for example, using a program such as ALIGN, which is accessible on the Internet (for example at http://www.hgsc.bcm.tmc.edu/SearchLauncher/). The mutations in the amino acid sequence are preferably so-called conservative changes, such as, for example, aspartic acid to glutamic acid or leucine to isoleucine. The specific binding can be demonstrated by standard tests, such as ELIS A.

In a further embodiment, a polypeptide in the sense of the present invention contains, in addition to FR-1, FR-2, FR-3, FR-4, CDR-1 and / or CDR-2, further constituents of an immunoglobulin, these constituents being natural, partly synthetic or can be of fully synthetic origin. Examples are components of the immunoglobulin isotypes, as well as parts of these immunoglobins, such as the constant chain components (C _H and / or QL) or parts thereof. Depending on the further constituents added, the polypeptides according to the invention can form Fab, F (ab *) ₂ , “single chain Fv” (scFv), Fv dAb or Fd fragments.

The term polypeptide is used for amino acid chains according to the invention of 9 or more amino acids. Polypeptides that have two or more identical ones

Have binding sites, have an increased functional affinity (= binding starch) and are therefore preferred embodiments of the polypeptide according to the invention. Polypeptides with increased affinity for endoglin can, for example, in the form of a diabody (= scFv dimer) (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90, 6444-6448), as a single-chain, multi-antigen-binding molecule (Brusselsbach et al., 1999, Tumor Targeting 4, 115-123), as tandem scFv or in fusion with dimerizing regions of immunoglobulins or dimerizing peptides and regions of other proteins (Plückthun & Pack, 1997, Immunotechnology 3 , 83-105).

A preferred embodiment of the polypeptide of the present invention is a polypeptide which contains one or more amino acid domains with a sequence according to SEQ ID No. 3. An amino acid domain in the sense of the present invention preferably has a length between approximately 80 and approximately 150 amino acids, more preferably between approximately 100 and approximately 120 amino acids and contains, in addition to a sequence according to SEQ ID No. 1, sequences of the human V _L region.

In a further embodiment, the polypeptide of the invention contains one or more amino acid domains with a sequence according to SEQ ID No. 4.

In a particularly preferred embodiment, the polypeptide in each case contains at least one amino acid domain according to SEQ ID No. 3 and at least one according to SEQ ID No. 4.

Preferably in the polypeptide according to the invention is between one or more amino acid domains according to SEQ ID No. 3 or SEQ ID No. 3 and SEQ ID No. 4 or between the one or more sequences according to SEQ ID No. 1 or SEQ ID No. 1 and SEQ ID No. 2 arranged a peptide linker. This peptide linker preferably has a length of approximately 12 to approximately 25 amino acids, in particular a length of 12 to 16 amino acids. The peptide linker is used for spatial separation of the domains and / or sequences and facilitates binding to Endoglin. A peptide linker with a sequence according to SEQ ID No. 5 is particularly suitable for the separation of the amino acid domains.

In a preferred embodiment, the polypeptide contains one or more secretion signals in order to facilitate the production and isolation of the polypeptide, which is expressed recombinantly, for example, in a suitable cell. By means of these secretion signals, the polypeptide according to the invention is excreted from the cell into the periplasm and can be obtained directly from the culture medium of the production cell line. The pelB secretion signal sequence (Lei et al., 1987, J. Bacteriol. 169, 4379-4383) with a sequence according to SEQ ID No. 6 is particularly suitable as the secretion signal.

In a further embodiment, the secretion signal sequences can be split off. This can be done, for example, by inserting peptide sequences that are recognized and cleaved by endopeptidases, or by inserting, for example, intein. The cleavage of the secretion signal sequences can be advantageous if the secretion signal sequences are immunogenic in humans and the cleavage of the sequences reduces the immunogenicity of the amino acid sequence according to the invention.

A particularly preferred polypeptide contains a sequence according to SEQ ID No. 7. Here, the pelB secretion signal sequence is arranged N-terminal, with the amino acid domains according to SEQ ID No. 3, the peptide linker according to SEQ ID No. 5 and the direction of the C-terminus Amino acid domains are arranged according to SEQ ID No. 4. In addition, the polypeptide also contains a hexahistidyl sequence (6 x His day) (Hochuli et al., 1988, Bio / Technol. 6, 1321-1325, Hoffmann & Roeder, 1991, Nucl. Acids Res., 19, 6337-6338) , which allows the purification of the antibody via, for example, a Ni ²⁺ affinity column, and a peptide which is recognized by the anti-Myc antibody 9E10 (Munro & Pelham, 1986, Cell 46, 291-300). Another object of the present invention is a polypeptide which contains a variant of the amino acid sequence according to SEQ ID No. 1. A variant of the sequence according to SEQ ID No. 1 are sequences in which two amino acids are deleted, one amino acid is deleted and one amino acid is mutated or two amino acids are mutated. Preferably, a mutation is even more preferred. Both mutations are so-called "conservative" mutations. One speaks of "conservative" mutations when an amino acid is replaced by an amino acid from the same class. The different amino acid classes are amino acids with non-polar, aliphatic side chains (Gly, Ala, Val, Leu, He and Pro), with polar, uncharged side chains (Ser, Thr, Cys, Met, Asn and Gin), with aromatic side chains (Phe , Tyr and Trp), with positively charged side chains (Lys, Arg and His) and with negatively charged side chains (Asp and Glu). Substitutions in which an amino acid is replaced by an amino acid with similar steric requirements, for example Ser against Thr or Gly against Ala, are particularly preferred within a class.

However, variants of the amino acid sequence according to SEQ ID No. 1 in which only one amino acid has been deleted or mutated are even more preferred. This mutation in the sequence according to SEQ ID No. 1 is preferably a conservative mutation.

The mutated and / or deleted polypeptides of the present invention are characterized in that they bind specifically to the extracellular domain of the enddoglin. This specific binding can be detected in ELISAs against immobilized endoglin or by precipitation of endoglin by the peptides according to the invention.

The polypeptide according to the invention can also be fused to at least one peptide and / or a protein. Amino acid sequences of less than 50 amino acids are referred to as peptides and amino acid sequences of 50 or more amino acids are referred to as proteins. A fusion occurs when the amino acids of the polypeptide have a peptide bond with the peptide and / or are linked to the protein. The fusion protein is preferably translated contiguously and encoded by an mRNA. Suitable proteins and peptides are, for example, enzymes, growth factors, hormones, cytokines, chemokines, viral coat proteins, and / or antibodies. The fusion with a cytokine or chemokine allows, for example, the recruitment of a substance that is toxic to the target cell and thus enables the targeted destruction of, for example, tumor endothelial cells. The fusion with a viral coat protein allows the production of recombinant viruses that carry a polypeptide on their surface that is specific for endoglin and thus the recruitment of the respective recombinant virus to endothelial cells. A suitable coat protein is, for example, the adenovirus fiber protein. A similar goal can also be achieved by fusion with an antibody, preferably with an scFv fragment, if this fragment specifically binds to a particular virus. Peptides or proteins that are recognized by viral surface molecules or an antibody are also suitable.

In a preferred embodiment of the polypeptide according to the invention, the protein or peptide binds specifically to a receptor. Specific binding can be detected, for example, on immobilized receptors with labeled polypeptides. Markings known in the prior art are, for example, radioactive markings or fluorescent markings. Suitable receptors are, for example, receptors that are present on cells of the immune system such as CD3, CD4, CD8 CD28, F _c α-1 receptor, F _c γ-1, 2 or 3 receptor. These cells can be recruited through interaction with endothelial cells.

Another object of the present invention is a polypeptide which is coupled to at least one component. Coupling means the covalent or non-covalent binding of a component to the polypeptide, the polypeptide and the component not being translated together and not being encoded by an mRNA. A covalent coupling between the invented polypeptide according to the invention and the component can be carried out, for example, by formaldehyde or glutaraldehyde. A non-covalent coupling is obtained, for example, by incubating a polypeptide according to the invention, which is fused with a peptide or protein which specifically binds to the knob domones of the adenoviral fiber protein, with adenovirus. Preferred components are peptides, proteins, enzymes, growth factors, hormones, cytokines, chemokines, viral coat proteins, carbohydrates, antibodies, lipids, isotopes, liposomes, viruses, virus-like particles, nucleic acids, and / or cells. The nucleic acids which are coupled to the polypeptide according to the invention can be “naked” or, for example, condensed with poly-lysine.

The coupling of the polypeptide according to the invention to liposomes is a particularly preferred embodiment of the present invention, since liposomes can be loaded with a wide variety of therapeutically active substances. Suitable liposomes are known for example from EP 0 555 333 or WO 00/74646. Preferred liposomes are anionic liposomes which contain an anionic phospholipid in addition to cholesterol. The ratio between cholesterol and phospholipid in the liposome is in the range between approximately 0.3 and approximately 1.2, preferably between approximately 0.4 and approximately 0.8. The polypeptide according to the invention is coupled to liposomes, for example via N-carboxylphosphatidylethanolamine or glutarylphosphatidylethanolamine.

The liposomes preferably contain at least one antisense RNA, at least one chemotherapeutic agent, at least one nucleic acid coding for an active substance or at least one active substance. If the liposomes contain nucleic acids, in a preferred embodiment the liposome additionally contains phosphatidylethanolamine (PEI), the PEI preferably having low molecular weight PEI with a molecular weight in the range from about 500 to about 25,000 Da, more preferably in a range from about 5,000 to 10,000 there is. The antisense RNA can, for example, inhibit the translation of genes that are required for cell division. Chemotherapy drugs include substances such as for example doxirubicin, cyclophosphamide, 5-fluorouracil, cis-platinum or taxol. The person skilled in the art is aware of further chemotherapeutic agents which are used in tumor therapy and which are encompassed by the present invention. An active ingredient that is encoded by a nucleic acid contained in the liposome can, for example, be an inhibitor of cell proliferation. Suitable proteins are known to the person skilled in the art and include anioncogens, such as, for example, p53 or pRb, and cell cycle inhibitors, such as, for example, p21 ^w ^ 16 ^, p57, p27 ^KIP or GADD45. Furthermore, the nucleic acids can also code for cytostatic or cytotoxic proteins, such as for example perforin, granzyme, IL-2, IL-4, IL-12 or Oncostatin M. An active substance can be any pharmacologically active substance which is suitable, for example, for the therapy of diseases in which endothelial cells are involved.

In a preferred embodiment of the polypeptide according to the invention, the component specifically binds to a receptor. A specific binding can be detected, for example, on immobilized receptors with labeled polypeptides and / or labeled components. Labels known in the prior art are, for example, radioactive labels or fluorescent labels. Suitable receptors are, for example, receptors that are present on cells of the immune system such as CD3, CD4, CD8 CD28, F _c α-1 receptor, F _c γ-1, 2 or 3 receptor. These cells are recruited by the interaction of the component with one of the cell surface proteins and by the interaction of the polypeptide according to the invention with endoglin to form endothelial cells.

Another object of the present invention is a nucleic acid which codes for a polypeptide according to the invention. It is known that there may be small changes in the sequence of a nucleic acid, for example due to the degeneracy of the genetic code, or that untranslated sequences may be attached to the 5 'and / or 3' end of the nucleic acid, without changing the encoded polypeptide. This invention therefore also includes so-called “variants” of the nucleic acids described above.

Variants of the nucleic acids are understood to mean all nucleic acid sequences that are complementary to a nucleic acid sequence that hybridize under stringent conditions with the reference sequence and that code for proteins that bind specifically to human endoglin.

“Stringent hybridization conditions” are to be understood as those conditions in which hybridization takes place at 60 ° C. in 2.5 × SSC buffer, followed by several washing steps at 37 ° C. in a lower buffer concentration and remains stable.

In order to enable the introduction of said nucleic acid and thus the expression of the polypeptide in a eu or prokaryotic cell by transfection, transformation or infection, the nucleic acid can be present as a plasmid, as part of a viral or non-viral vector.

Another object of the present invention is therefore a vector, in particular an expression vector, which contains a nucleic acid coding for a polypeptide according to the invention. Baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses are particularly suitable as viral vectors. Virosomes, liposomes, cationic lipids or poly-lysine-conjugated DNA are particularly suitable as non-viral vectors.

The present invention furthermore relates to a cell which contains at least one nucleic acid according to the invention and / or at least one vector according to the invention. This cell expresses the polypeptide according to the invention under conditions known to the person skilled in the art which lead to the activation of the regulatable elements used in each case. The polypeptide can then be derived from the Cell are isolated or is secreted by the cell. Procaryotic and eukaryotic cells, in particular bacterial cells such as E. coli, yeast cells such as S. cerevisiae, insect cells such as Spodoptera frugiperda cells (Sf-9) or Trichoplusia ni cells are suitable for the genetic engineering production and subsequent purification of the expressed compounds according to the invention or mammalian cells such as COS cells or HeLa cells.

Another object of the present invention is therefore a method for producing a polypeptide according to the invention, in which at least one nucleic acid according to the invention is expressed in a cell. If the polypeptide according to the invention contains a cleavable secretion signal sequence, this can be cleaved in a further step, for example by incubation with a suitable endopeptidase or, in the case of intein, by adding dithiothreitol (DTT) to the medium.

If a component is to be coupled to the polypeptide according to the invention, this coupling can take place by incubation or chemical reaction with at least one component. Such a coupling can preferably already take place in the cell only after the polypeptide has been purified.

The polypeptide according to the invention can be used as a diagnostic tool. Another object of the present invention is accordingly the use of at least one polypeptide for the detection of endoglin and / or endoglin-expressing cells or cell components in vitro and / or in vivo.

Detection can be carried out directly by fusion or coupling of a detectable component (for example with an enzyme or a radioisotope) or indirectly by means of a labeled component which recognizes the polypeptide according to the invention. Detection methods used with preference are ELISA, RIA, immunofluorescence, immunoprecipitation or immunoscintillation. The polypeptide according to the invention directed against endoglin can also serve as a ligand in order to specifically recognize and bind endoglin-expressing cells (eg tumor endothelial cells). Another object of the present invention is accordingly the use of at least one polypeptide according to the invention for binding to endoglin-expressing cells.

By connecting to a second ligand by coupling or fusion, at least one peptide, at least one protein or at least one component can be recruited to endoglin-expressing cells. This second ligand can be an antibody molecule or fragment, a ligand for a cellular receptor, a peptide that recognizes a receptor on cells.

In a preferred use, the polypeptide according to the invention has a cytotoxic effect on the endoglin-expressing cell. This effect is achieved, for example, by recruiting cytotoxic T cells, fusing or coupling with cytokines or enzymes, such as “prodrug converting enzymes”.

In a further use of the polypeptide according to the invention, the binding to the endoglin-expressing cell leads to infection, transduction or transfection of the cell with a virus, a Vims-like particle, a liposome and / or a nucleic acid.

Another object of the present invention is the use of at least one polypeptide, at least one nucleic acid and / or at least one vector as described above for the therapy of diseases in which endothelial cells are involved. In a preferred embodiment, the polypeptides, nucleic acid and / or vectors according to the invention are used for the therapy of diseases which are characterized by the hyperproliferation of endoglin-expressing cells. Hyperproliferation of endo- For example, cell cells are observed in the neovascularization of tumor tissue, which is why the therapy of tumor diseases is a particularly preferred use of the polypeptides, nucleic acids and / or vectors according to the invention.

Another object of the present invention is a medicament or diagnostic that contains at least one polypeptide, at least one nucleic acid, and / or at least one vector as described above and optionally suitable hoof and additives. Suitable auxiliaries and additives, for example, improve the shelf life and improve the tolerability or increase the availability of the medicament or diagnostic agent according to the invention and are known to the person skilled in the art.

The following figure and the following examples are only intended to describe the invention in more detail without restricting it.

Fig. 1: The DNA sequence and derived protein sequence of the anti-endoglin polypeptide C4 according to the invention in the form of an scFv fragment. The signal sequence, the connecting peptide and the C-terminal sequences for cleaning and detection are underlined. The individual nucleotide areas have the following meaning:

Nucleotides 1-42 5 'untranslated region

Nucleotides 43-106 DNA coding for pelB signal sequence (Lei et al., 1987, J. Bacteriol. 169, 4379-4383)

Nucleotides 107-465 DNA coding for human VH domain (semisynthetic consists of germ line V gene and synthetic CDR3-

FR4 region) (Griffiths et al., 1994, EMBO J. 13, 3245-

3260) nucleotides 466-505 DNA coding for artificial peptide sequence (Huston et al.,

1988) Nucleotides 506-828 DNA coding for human VL domain (semisynthetically consists of germ line V gene and synthetic CDR3-FR4 region) (Griffiths et al., 1994, EMBO J. 13, 3245-3260) coding nucleotides 829-837 DNA for artificial peptide sequence

Nucleotides 838-855 DNA coding for hexahistidyl sequence (Hochuli et al.,

1988, Bio / Technol. 6, 1321-1325)

Nucleotides 856-864 DNA coding for artificial peptide sequence

Nucleotides 865-897 DNA coding for epitope of the anti-Myc antibody 9E10 (Munro & Pelham, 1986, Cell 46, 291-300)

Nucleotides 898-906 DNA coding for artificial peptide sequence

Examples

Example 1: Detection of endoglin on primary endothelial cells

The polypeptide scFv C4 shown in Fig. 1, which was isolated by phage display (Kontermann & Dübel 2000, Antibody Engineering, Springer Verlag), which was present in the expression plasmid pHEN2 (MRC Center for Protein Engineering, Cambridge, UK) was purified after the induction of protein expression by adding isopropyl-β-D-galactopyranoside (IPTG) from periplasmic extracts of TG1 bacteria by means of immobilized metal affinity chromatography. For this, 10 ml of an overnight culture of scFv C4 were added per liter of LB medium which had been admixed with 100 μg / ml ampicillin and 0.1% glucose and shaken at 37 ° C. When an OD ₆₀ o of 0.8 was reached, a final concentration of 1 mM was added and the bacteria were shaken at room temperature for 3 hours. The bacteria were centrifuged off and the pellet was resuspended with extraction buffer (30 mM Tris-HCl pH 8, 1 mM EDTA, 20% sucrose). After an incubation of 15 min on ice, MgCl _{2 was added} to a final concentration of 5 mM and the solution was centrifuged again. The supernatant was dialyzed against IMAC loading buffer (50 mM sodium phosphate buffer pH 7.5, 500 mM NaCl, 20 mM imidazole). The dialysate was loaded onto a Ni-NTA-loaded column (Qiagen), which was equilibrated with loading buffer, washed with washing buffer (50 mM sodium phosphate buffer pH 7.5, 500 mM NaCl, 35 mM imidazole) and the bound antibody fragment subsequently with elution buffer (50 mM sodium phosphate buffer pH 7.5, 500 mM NaCl, 100 mM imidazole) eluted.

The purified polypeptide was used to detect endoglin. The bound polypeptide scFv C4 was detected indirectly with the help of monoclonal antibodies, which were either directed against the hexahistidyl sequence or against the Myc epitope. Binding to purified endoglin was demonstrated by means of ELISA. For this purpose, a polystyrene microtite latte was coated with human endoglin (conc. 10 μg / ml in PBS) overnight at 4 ° C. After a washing step in PBS, free binding sites were saturated by incubation with PBS, 2% skim milk powder. The anti-endoglin antibody was adjusted to a concentration of 50 μg / ml - 5 ng / ml in PBS, 2% skim milk powder, 100 μl / well each was added to the microtiter plate and incubated for 1 hour at room temperature. The plate was then washed with PBS for 5 min. Bound antibodies were detected with a peroxidase-labeled second antibody that recognizes the C-terminal Myc-tag of scFv C4. The second antibody was adjusted to a concentration of 1 μg / ml in PBS and 100 μl was added to each well of the microtiter plate. After incubation for 1 hour at room temperature, washing was again carried out for 5 minutes with PBS. Bound antibodies were detected by conversion of the peroxidase substrate tetrametylbenzidine / HO ₂ . After adding 50 μl of 1 M sulfuric acid, the color change was determined in a photometer at a wavelength of 450 nm.

Endoglin from primary human umbilical cord endothelial cells (HUVEC) was detected by immunoprecipitation under non-denaturing conditions. rejected. For this purpose, the [S] -methionine-labeled endothelial cells were lysed with lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P40) for 30 min at 4 ° C. After an ultracentrifugation step at 40,000 rpm for 20 min, the supernatant was mixed with 5 μg scFv C4, 5 μg of a negative control scFv or 5 μl of the murine anti-endoglin antibody SN6h (REF) and incubated for 1 hour at 4 ° C. This was followed by incubation with the anti-Myc antibody 9E10 (Munro & Pelham, 1986, Cell 46, 291-300) and then with Protein A-Sepharose, each for 30 min at 4 ° C. The complexes were washed several times with lysis buffer and finally resuspended in 20 μl SDS-PAGE loading buffer. After separation in the SDS polyacrylamide gel, the gel was fixed in 30% methanol and 10% acetic acid for 30 min and then amplification solution was added (Amersham-Buchler). The gel was dried and exposed to an X-ray film.

It was shown that the polypeptide scFv C4 specifically precipitated a band which was identical to that of the murine anti-endoglin antibody SN6h, while this band was not detectable with the negative control antibody. Thus, scFv C4 specifically detected endoglin in extracts from primary endothelial cells.

In further experiments, endoglin was detected on cells using immunofluorescence. For this purpose, various endothelial cells (HUVEC, HMVEC, HDMEC, HMEC) and non-endothelial cells (A549, HEK293) were incubated with scFv C4 with a concentration of 5 - 25 μg / ml or with the negative and the positive control antibody for 30 min at 4 ° C , The recombinant polypeptides were then incubated with the anti-Myc antibody 9E10 for 30 min at 4 ° C. Finally, all batches were incubated with a Cy3-labeled anti-mouse antibody. The bound polypeptides were detected either by means of fluorescence microscopy or flow cytometry. In these experiments, specific fluorescence from endoglin-expressing endothelial cells could be detected, while various enddoglin-negative cells showed no reaction. This showed a typical surface staining of the cells, as was to be expected for a membrane protein.

Example 2: A bispecific single-chain, multi-antigen binding molecule for a targeted transduction of endothelial cells with adenoviruses

The construction of a bispecific single-chain, multi-antigen-binding molecule (reference is also made to patent application DE 198 16 141 and EP 0 952 218), which is directed against endoglin and the knob domain of the fiber protein of adenoviruses of serotype 5, was carried out at the DNA level as follows. The scFv fragment S11 was used for this (Watkins et al., 1996, Gene Ther. 4: 1004-1012). ScFv Sl l binds to the knob domain of the fiber protein and neutralizes wild-type infection through this binding. A polymerase chain reaction was used to attach to the V _L fragment of scFv Sl l sequences which at the 5 'end for a BstEII restriction endonuclease interface and a five amino acid connecting peptide and at the 3' end eight amino acids of the middle connecting peptide and an AscI Encode restriction endonuclease interface. In the same way, sequences were attached to the Vπ fragment of scFv S1, those at the 5 'end for seven amino acids of the middle connecting peptide and an AscI-Res endonuMease interface and at the 3' end for a SacI restriction endonuclease interface and one five amino acid encode long connecting peptide. These fragments were cloned into the plasmid pABl-scFv C4. The resulting bispecific single-chain multi-antigen-binding molecule (EDG-Ad) has the structure VHC4-PeptideA-VLS11-PeptideMNHSII-PeptideB-VLC4. Peptide A and B each have the sequence GGGGS and peptide M the sequence GGGGSGGRASGGGGGS. The monomeric molecule has a molecular weight of about 58 kDa and has a binding site for Endoglin and the knob domain. The bispecific single-chain, multi-antigen binding molecule was purified from the periplasm of induced bacteria as described in Example 1. Binding studies showed that this molecule was fully functional. It recognized the knob domain in ELISA and endoglin-expressing HUVEC in immunofluorescence.

In order to investigate the adenoviral transduction, 2 x 10 ^ HUVECs or 3.5 x 10 ^ A549 cells were plated on 96-well plates two days before infection with viruses. AdCMVLacZ, which expresses the lacZ gene under the control of the CMV promoter, were incubated with the bispecific single-chain, multi-antigen-binding molecule EDG-Ad for 1 hour at 37 ° C. and then added to the cells for 1 hour. Viruses that were not incubated with EDG-Ad served as a control. The β-galactosidase was expressed by means of X-Gal staining. For this, the cells were fixed after a PBS washing step with 0.1% glutaraldehyde, washed again with PBS and then with 0.8 mg / ml X-Gal, 3 mM K3Fe (CN) 6, and 3 mM K-4Fe ( CN) 6 incubated in PBS at 37 ° C.

These experiments showed that adenoviruses alone showed only a very weak transduction with the virus titer used (8 x 10 ⁵ pfu). However, this was significantly increased by complexing with EDG-Ad. This increased transduction mediated by EDG-Ad was dependent on the presence of Endoglin on the target cells. Endoglin-negative cells (eg A549) were not transduced to an increased extent. EDG-Ad-mediated transduction of HUVEC was also inhibited by preincubation with scFv C4. In contrast, the soluble knob domain, which completely inhibits wild-type transduction by binding to the primary receptor (Coxsackie adenovirus receptor CAR), had no influence on EDG-Ad-mediated transduction. Pre-incubation with an RGD peptide, which inhibits the interaction of the adenoviral penton base with the secondary receptor (α _v integrin) and thereby also the wild type transduction prevented, also had no influence on the EDG-Ad-mediated transduction. EDG-Ad-mediated transduction of endoglin-expressing cells is therefore independent of the presence of the adenoviral receptors. The EDG-Ad-mediated transduction takes place rather directly or indirectly via Endoglin. The results show that it is possible to use a bispecific molecule, which is directed against endoglin and a Vimshüllprotein, to specifically recruit viruses to endoglin-expressing endothelial cells.

Example 3: A bispecific single-chain, multi-antigen-binding molecule for a targeted solution of endothelial cells by cytotoxic T-lymphocytes

The construction of a bispecific single-chain, multi-antigen-binding molecule (reference is also made here to patent application DE 198 16 141 and EP 0 952 218), which is directed against endoglin and the ε chain of the T-cell receptor CD3, was carried out on DNA Level as follows. For this purpose, scFv CD3v9 was used, which binds to the ε chain of the T cell coreceptor CD3. ScFv CD3v9 is a humanized antibody fragment of the monoclonal antibody UCHT1 (Zhu & Carter, 1995, J. Immunol. 155: 1903-1910). A polymerase chain reaction was used to attach to the V _L fragment of scFv CD3 sequences which at the 5 'end for a BstEII restriction endonuclease interface and a five amino acid connecting peptide and at the 3' end eight amino acids of the middle connecting peptide and an AscI restriction endonuclease interface encode. In the same way, sequences were attached to the Vπ fragment of scFv CD3, which at the 5 'end for seven amino acids of the middle connecting peptide and an AscI restriction endonuclease interface and at the 3' end for a Sacl restriction endonuclease interface and a five amino acids connecting compound coding peptide. These fragments were cloned into the plasmid pABl-scFv C4. The resulting bispecific single chain multi-antigen binding Molecule (EDG-CD3) has the structure VHC4-PeptideA-VLCD3-PeptideMNHCD3-PeptideB-VLC4. Peptide A and B each have the sequence GGGGS and peptide M the sequence GGGGSGGRASGGGGGS. The monomeric molecule has a binding site for endoglin and CD3. The bispecific single-chain, multi-antigen-binding molecule was purified from the periplasm of induced bacteria as described in Example 1. Binding studies showed that this molecule was fully functional. It recognized both Endoglin-expressing HU-VECs and CD3-expressing Jurkat cells in immunofluorescence.

Europium-labeled HUVECs and isolated human T-lymphocytes activated by phytohemagglutinin and IL-2 were used for the analysis of an EDG-CD3 -mediated cytolysis of endothelial cells by cytotoxic T-lymphocytes. These cells were in a ratio of HUVECs (target cell) to T-lymphocytes (effector) of 1: 3, 1: 10, 1:30 and 1: 100 with different concentrations of EDG-CD3 (10 μg / ml to 1 ng / ml). After an incubation of 4 hours in the cabinet, the lysis of the endothelial cells was measured using time-resolving fluorescence. These results showed EDG-CD3-dependent cytolysis of the HUVECs. This was strongest at EDG-CD3 concentrations between 1-10 μg / ml and an effector-target cell ratio of 100. Experiments with endoglin-negative control cells as well as the use of a bispecific single-chain, multi-antigen-binding molecule that was effective against EDG and ß-galactosidase ( EDG-Gal) showed no lysis of the endothelial cells. These experiments demonstrate that EDG-CD3 is able to recruit T cells to endoglin-expressing endothelial cells and thereby trigger cell lysis.

Claims

claims 1. Polypeptide that binds specifically to the extracellular domain of human endoglin (CD 105) protein, characterized in that the polypeptide contains one or more sequences as shown in SEQ ID No. 1. 2. Polypeptide according spoke 1, characterized in that the polypeptide contains one or more sequences according to SEQ ID No. 2. 3. Polypeptide according spoke 1, characterized in that the polypeptide contains one or more amino acid domains of a human antibody, these amino acid domains are selected from the framework region-1 (FR-1), FR-2, FR-3, FR-4, the. complementarity determining region- 1 (CDR-1) and CDR-2 of the antibody. 4. Polypeptide according to one of claims 1 to 3, characterized in that the polypeptide contains one or more amino acid domains with a sequence according to SEQ ID No. 3. 5. Polypeptide according to one of claims 1 to 4, characterized in that the polypeptide contains one or more amino acid domains with a sequence according to SEQ ID No. 4 6. Polypeptide according to one of claims 3 to 5, characterized in that a peptide linker is arranged in each case between the amino acid domains. 7. Polypeptide according spoke 6, characterized in that the peptide linker contains a sequence according to SEQ ID No. 5. 8. Polypeptide according to one of claims 1 to 7, characterized in that the polypeptide contains one or more secretion signal sequences. 9. Polypeptide according to spoke 8, characterized in that the secretion signal sequence contains a sequence according to SEQ ID No. 6. 10. Polypeptide according to one of claims 1 to 9, characterized in that the polypeptide contains one or more sequences according to SEQ ID No. 7. 11. Polypeptide according to one of claims 1 to 10, characterized in that the polypeptide contains a variant of SEQ ID No. 1. 12. Polypeptide according to one of claims 1 to 11, characterized in that the polypeptide is fused with at least one peptide or protein. 13. Polypeptide according to claim 12, characterized in that the protein or the peptide, an enzyme, a growth factor, a hormone, a cytokine, a chemokine, a viral coat protein, and / or an antibody. 14. Polypeptide according to spoke 12 or 13, characterized in that the protein or the peptide is able to bind specifically to a receptor. 15. Polypeptide according to one of claims 1 to 14, characterized in that the polypeptide is coupled to at least one component. 16. Polypeptide according to spoke 15, characterized in that the component is a peptide, a protein, an enzyme, a growth factor, a hormone, a cytokine, a chemokine, a viral coat protein, a carbohydrate, an antibody, a lipid, an isotope, is a liposome, a virus, a virus-like particle, a nucleic acid, and / or a cell. 17. Polypeptide according to spoke 15 or 16, characterized in that the component is able to bind specifically to a receptor. 18. Polypeptide according spoke 17, characterized in that the liposome contains at least one antisense RNA, at least one nucleic acid coding for an active ingredient or at least one active substance. 19. Polypeptide according to spoke 18, characterized in that the active substance is a chemotherapeutic agent 20. Nucleic acid, characterized in that the nucleic acid codes for a polypeptide according to one of claims 1 to 14. 21. Vector, characterized in that the vector contains at least one nucleic acid according to claim 20. 22. Cell, characterized in that the cell contains at least one nucleic acid according to claim 20 and / or at least one vector according to claim 21. 23. A method for producing a polypeptide according to any one of claims 1 to 14, characterized in that at least one nucleic acid according to claim 20 is expressed in a cell. 24. The method according to claim 23, characterized in that in a further step at least one component is coupled to the polypeptide. 25. Use of at least one polypeptide according to one of claims 1 to 19 for the detection of endoglin and / or endoglin-expressing cells or cell components in vitro and / or in vivo. 26. Use according to claim 25, characterized in that the detection is carried out with an ELISA, a RIA, an immunofluorescence, an immunoprecipitation or an immunoscintillation. 27. Use of at least one polypeptide according to one of claims 1 to 19 for binding to endoglin-expressing cells, characterized in that the binding of the polypeptide has a cytotoxic effect on the endoglin-expressing cell. 28. Use of at least one polypeptide according to one of claims 1 to 19, for infection, transduction or transfection of endoglin-expressing cells. 29. Use of at least one polypeptide according to one of claims 1 to 19, at least one nucleic acid according to claim 20 and / or at least one vector according to claim 21 and for the manufacture of a medicament for Diagnosis and / or therapy of a disease characterized by hyperproliferation of endoglin-expressing cells. 30. Use according to claim 29, characterized in that the disease is a tumor rhinoceros. 31. Medicament or diagnostic agent containing at least one polypeptide according to one of claims 1 to 19, at least one nucleic acid according to claim 20, and / or at least one vector according to claim 21 and optionally suitable auxiliaries and additives.