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EP1315760A2 - Polypeptide specifique de l'endogline, sa fabrication et son utilisation - Google Patents

Polypeptide specifique de l'endogline, sa fabrication et son utilisation

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Publication number
EP1315760A2
EP1315760A2 EP01980336A EP01980336A EP1315760A2 EP 1315760 A2 EP1315760 A2 EP 1315760A2 EP 01980336 A EP01980336 A EP 01980336A EP 01980336 A EP01980336 A EP 01980336A EP 1315760 A2 EP1315760 A2 EP 1315760A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
endoglin
polypeptide according
seq
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Withdrawn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
EP01980336A
Other languages
German (de)
English (en)
Inventor
Roland Kontermann
Daniel Miller
Rolf Müller
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.)
PHARMEXA AS
Original Assignee
Vectron Therapeutics AG
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 Vectron Therapeutics AG filed Critical Vectron Therapeutics AG
Publication of EP1315760A2 publication Critical patent/EP1315760A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Endoglin specific polypeptide 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).
  • 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.
  • 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.
  • receptors are, for example, the VEGF receptors or the ⁇ v integrins (Burrows & Thorpe, 1994, Pharmac. Ther. 64, 155-174).
  • Antibody fragments that recognize specific structures in the vascular bed can also be used.
  • 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).
  • 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.
  • HAMA human anti-mouse antibodies
  • 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.
  • 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 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.
  • a peptide linker is inserted between every two sequences. The peptide linker is preferably between about 12 and about 25 amino acids in length.
  • the structure of the CDRs determines the binding specificity of the antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polypeptides according to the invention can form Fab, F (ab *) 2 , “single chain Fv” (scFv), Fv dAb or Fd fragments.
  • 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
  • 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.
  • polypeptide of the invention contains one or more amino acid domains with a sequence according to SEQ ID No. 4.
  • polypeptide linker 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.
  • 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.
  • secretion signals 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.
  • 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.
  • 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.
  • the polypeptide also contains a hexahistidyl sequence (6 x His day) (Hochuli et al., 1988, Bio / Technol. 6, 1321-1325, Hoffmann & Roeder, 1991, Nucl.
  • 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.
  • 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.
  • 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.
  • PEI phosphatidylethanolamine
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • the nucleic acid can be present as a plasmid, as part of a viral or non-viral vector.
  • 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.
  • 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.
  • DTT dithiothreitol
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 107-465 DNA coding for human VH domain (semisynthetic consists of germ line V gene and synthetic CDR3-
  • nucleotides 466-505 DNA coding for artificial peptide sequence (Huston et al.,
  • 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.,
  • Example 1 Detection of endoglin on primary endothelial cells
  • IPTG isopropyl- ⁇ -D-galactopyranoside
  • 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.
  • a polystyrene microtite latte was coated with human endoglin (conc. 10 ⁇ g / 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 ⁇ g / ml - 5 ng / ml in PBS, 2% skim milk powder, 100 ⁇ l / 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 ⁇ g / ml in PBS and 100 ⁇ l 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 ⁇ l 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 was detected by immunoprecipitation under non-denaturing conditions. rejected.
  • 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.
  • the supernatant was mixed with 5 ⁇ g scFv C4, 5 ⁇ g of a negative control scFv or 5 ⁇ l 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 ⁇ l 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.
  • 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.
  • scFv C4 specifically detected endoglin in extracts from primary endothelial cells.
  • endoglin was detected on cells using immunofluorescence.
  • various endothelial cells HMVEC, HDMEC, HMEC
  • non-endothelial cells A549, HEK293
  • scFv C4 a concentration of 5 - 25 ⁇ g / 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.
  • 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
  • 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.
  • sequences were attached to the V ⁇ 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.
  • the resulting bispecific single-chain multi-antigen-binding molecule 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.
  • 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.
  • 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.
  • 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.
  • sequences were attached to the V ⁇ 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.
  • 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.
  • EDG-CD3-dependent cytolysis of the HUVECs This was strongest at EDG-CD3 concentrations between 1-10 ⁇ g / ml and an effector-target cell ratio of 100.
  • EDG-Gal ß-galactosidase

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Abstract

L'invention concerne un polypeptide se liant spécifiquement au domaine extra-cellulaire de la protéine de l'endogline humaine (CD105), ainsi que sa fabrication et son utilisation.
EP01980336A 2000-09-04 2001-09-04 Polypeptide specifique de l'endogline, sa fabrication et son utilisation Withdrawn EP1315760A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10043481 2000-09-04
DE10043481A DE10043481A1 (de) 2000-09-04 2000-09-04 Humaner Antikörper gegen Endoglin (CD105) und seine Verwendung
PCT/EP2001/010197 WO2002020614A2 (fr) 2000-09-04 2001-09-04 Polypeptide specifique de l'endogline, sa fabrication et son utilisation

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EP1315760A2 true EP1315760A2 (fr) 2003-06-04

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US (1) US20040053329A1 (fr)
EP (1) EP1315760A2 (fr)
JP (1) JP2004508035A (fr)
CA (1) CA2421202A1 (fr)
DE (1) DE10043481A1 (fr)
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DE10043481A1 (de) 2002-04-11
US20040053329A1 (en) 2004-03-18
WO2002020614A2 (fr) 2002-03-14
WO2002020614A3 (fr) 2002-08-01
JP2004508035A (ja) 2004-03-18

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