EP0990034A1 - Anticorps recombines anti-gpiib/iiia - Google Patents

Anticorps recombines anti-gpiib/iiia

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Publication number
EP0990034A1
EP0990034A1 EP98934922A EP98934922A EP0990034A1 EP 0990034 A1 EP0990034 A1 EP 0990034A1 EP 98934922 A EP98934922 A EP 98934922A EP 98934922 A EP98934922 A EP 98934922A EP 0990034 A1 EP0990034 A1 EP 0990034A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
ser
gly
acid sequence
nucleotide sequence
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Withdrawn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
EP98934922A
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German (de)
English (en)
Inventor
Peter Berchtold
Robert F. A. Escher
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.)
ASAT AG Applied Science and Technology
Original Assignee
ASAT AG Applied Science and Technology
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Filing date
Publication date
Priority claimed from DE19820663A external-priority patent/DE19820663A1/de
Application filed by ASAT AG Applied Science and Technology filed Critical ASAT AG Applied Science and Technology
Publication of EP0990034A1 publication Critical patent/EP0990034A1/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Withdrawn legal-status Critical Current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
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to new nucleic acid sequences which code for human autoantibodies against platelet membrane proteins and for anti-idiotypic antibodies, new amino acid sequences of human antibodies and their use for the diagnosis and therapy of diseases.
  • Autoimmune thrombocytopenic purpura is an immune disease, which is defined by a low platelet count in normal or increased megakaryocytopoiesis. Due to the presence of anti-platelet autoantibodies, there is an increased destruction of platelets in the reticuloendothelial system (spleen, liver, bone marrow). These autoantibodies, which can be detected in approximately 75% of AlTP patients, are mainly directed against the platelet membrane glycoproteins (GP) llb / llla and Ib / IX. Several different auto-antibody specificities can be found in a single patient (see e.g.
  • Antibodies Hybridomas 1 (1 990), 83-95 The occurrence of natural autoantibodies against various self-antigens has also been reported in healthy persons, for example against intracellular and cytoskeletal components of human platelets (Guilbert et al., J. Immunol. 1 28 (1 982), 2779-2787; Hurez et al., Eur. J. Immunol. 23 (1 993), 783-789 and Pfueller et al., Clin. Exp. Immunol. 79 (1 990), 367-373). Some of these autoantibodies observed in the serum of healthy people can also be directed against platelet membrane proteins (Souberbielle, Eur. J. Haematol. 56 (1 996), 1 78-1 80). The role of these natural autoantibodies and their relationship to disease-associated autoantibodies is still unknown.
  • Corticosteroids can be used to treat AlTP. Approximately half of the patients respond to prednisone administration within 4 weeks, but long-term remissions are rarely found. In patients who have heavy bleeding or extremely low platelet counts, the administration of high doses of intravenous immunoglobulin (IVIgG) is recommended as an emergency treatment. This treatment is followed by a rapid, but usually only temporary, increase in platelet counts in most patients. The mechanisms of action of corticosteroids and IVIgG in the treatment of AlTP are still unknown.
  • IVIgG intravenous immunoglobulin
  • the problem underlying the present application is to identify new DNA sequences which are responsible for the binding of autoantibodies to GPIIb / llla.
  • new pharmaceutical preparations can be provided, which can be used to improve the diagnosis and therapy of AlTP.
  • binding sequences from autoantibodies were identified after production of a combinatorial phagemid display library of heavy and light chains of human antibodies using peripheral circulating B cells from a healthy human donor. After presentation of human heavy and light antibody Fab fragments on the surface of the filamentous phage M 1 3, phage clones could be identified which show a specific binding to GPIIb / llla.
  • the phagemid library was successively brought into contact with platelet platelets without GPIIb / llla (negative selection) and normal platelets (positive selection). After several rounds of selection and amplification by infection of E. coli, 23 clones were obtained which can bind to the GPIIb / llla complex. Inhibition studies using pools of monoclonal antibodies to GPIIb / llla revealed two groups of clones: both groups were inhibited by monoclonal antibodies specific for the GPIIb / llla complex and one group also by a GPIIb specific monoclonal antibody.
  • the phage clones contain the antigen binding sequences of natural autoantibodies that come from the genome of a healthy person, this finding can lead to new insights into the origin of platelet-associated autoantibodies in AlTP.
  • using the phage clones according to the invention it is possible to generate recombinant anti-idiotypic antibodies against anti-GPIIb / Illa autoantibodies, the anti-GPIIb / IIla phage clones being used as the antigen.
  • the recombinant anti-idiotypic antibodies obtainable in this way represent an interesting clinical alternative to the use of IVIgG.
  • nucleotide and derived amino acid sequences of the identified phage clones are in the sequence listing SEQ ID No.1 to 8 (autoantibodies) and SEQ ID No. 9 to 1 8 (anti-idiotypic antibodies).
  • a first aspect of the present invention relates to nucleic acids which code for autoantibodies.
  • the invention thus relates to a nucleic acid which codes for the heavy chain of a human antibody, a functional derivative or a fragment thereof and which comprises a CDR3 region selected from: (a) one for the amino acid sequence:
  • nucleic acid preferably further comprises a CDR1 region selected from
  • (c) a nucleotide sequence which codes for an amino acid sequence with a homology of at least 80% and preferably at least 90% to an amino acid sequence from (a) or (b).
  • the nucleic acid according to the invention preferably further comprises a CDR2 region selected:
  • (c) a nucleotide sequence which codes for an amino acid sequence with a homology of at least 80% and preferably of at least 90% to an amino acid sequence from (a) or (b).
  • a second aspect of the present invention is a nucleic acid encoding a human antibody light chain, a functional derivative or a fragment thereof and comprising a CDR3 region selected from: (a) one for the amino acid sequence:
  • a TW DDGLNG PV (VII) coding nucleotide sequence (b) one for the amino acid sequence:
  • a AW DDSLNG WV (VIII) coding nucleotide sequence (c) a nucleotide sequence which codes for an amino acid sequence with a homology of at least 80% and preferably of at least 90% to an amino acid sequence from (a) or (b) and (d) one Nucleotide sequence coding for an amino acid sequence with an equivalent binding ability to GPIIb / llla.
  • the nucleic acid according to the invention preferably further comprises a CDR1 region selected from:
  • nucleic acid according to the invention preferably further comprises a CDR2 region selected from: (a) one for the amino acid sequence:
  • SNNQRPS (XII) coding nucleotide sequence and (c) a nucleotide sequence which codes for an amino acid sequence with a homology of at least 80% and preferably at least 90% to an amino acid sequence from (a) or (b).
  • a second aspect of the present invention relates to nucleic acids which code for anti-idiotypic antibodies.
  • An object of the invention is thus a nucleic acid encoding the heavy chain of a human antibody, a functional derivative or a fragment thereof and comprising a CDR3 region selected from:
  • GSGSYLGYYFDY (XX) coding nucleotide sequence, (i) one for the amino acid sequence:
  • XXI coding nucleotide sequence
  • j a nucleotide sequence which is suitable for an amino acid sequence with a homology of at least 80% and preferably of at least 90% to an amino acid sequence from (a), (b), (c) or (d) encodes and (k) a nucleotide sequence for an amino acid sequence with an equivalent binding ability to autoantibodies against
  • the nucleic acid according to the invention preferably further comprises a CDR1 region selected from: a nucleotide sequence coding for the amino acid sequences NFAMS, SYTMH, DYALH or SHYWS shown in Table 7a, a nucleotide sequence coding for the amino acid sequence TYYWS, one for the amino acid sequences DYGM shown in Table 7b , SHTIS, KYAIH or ELSMH coding nucleotide sequence and a nucleotide sequence which codes for an amino acid sequence with a homology of at least 80% and preferably at least 90% to one of the aforementioned amino acid sequences.
  • the nucleic acid according to the invention preferably further comprises a CDR2 region selected from one for the amino acid sequences GISGGG LLT H YA (D / N) SVK G, LI SY DGSN KY YADSVKG, GI SW D STS IGYAD SV KG or FIYDGART RFN PSLRS for the amino acid sequences shown in Tab coding nucleotide sequence, a nucleotide sequence coding for the amino acid sequence YIYYSGNTNYNPSLKS, one for the Amino acid sequences shown in Table 7b AI SY DGSNK YYA DSVKG, GITPIFG TV N YA QKF QG, AISSNGG NTYYA DSVKG or G FD PE DGE TIY AQ KF QG and a nucleotide sequence coding for an amino acid sequence with preferably at least 80% and a homology of at least 80% encoded by at least 90% to one of the aforementioned amino acid sequences.
  • the present invention further provides a nucleic acid which codes for the light chain of a human antibody, a functional derivative or a fragment thereof and which comprises a CDR3 region selected from:
  • the nucleic acid according to the invention preferably further comprises a CDR1 region selected from a nucleotide sequence coding for the amino acid sequence TGTS SAI G NYN FVP shown in Table 7a, a nucleotide sequence coding for the amino acid sequence GGYKIGSKSVH shown in Table 7b and a nucleotide sequence containing an amino acid sequence a homology of at least 80% and preferably encoded by at least 90% of the aforementioned amino acid sequence.
  • the nucleic acid according to the invention preferably further comprises a CDR2 region selected from a nucleotide sequence coding for the amino acid sequence shown in Table 7a EGSKRPS, a nucleotide sequence coding for the amino acid sequence shown in Table 7b and a nucleotide sequence for an amino acid sequence with a homology of at least 80% and preferably at least 90% to the amino acid sequence mentioned above.
  • the term "functional derivative of a chain of a human antibody” in the sense of the present invention is to be understood as a polypeptide which comprises at least one CDR3 region of the heavy and / or light chain as defined above and optionally together with the respective complementary chain of the human Antibody (or a derivative of such a chain) can form an antibody derivative that has an equivalent recognition specificity for an antigen as the non-derivatized antibody.
  • Such an antibody derivative preferably has a binding constant of at least 10 6 l / mol, preferably of at least 10 8 l / mol, for the respective antigen.
  • Functional derivatives of chains of a human antibody can be produced, for example, by deletion, substitution and / or insertion of sections of the gene coding for the respective polypeptide by recombinant DNA techniques.
  • antibody chains or antibodies are single-chain antibodies, which for example consist of the variable ones
  • H and L chain or one or two H chain domains and optionally a constant domain can be composed.
  • the construction of such constructs is described in Hoogenboom et al., Immunol. Rev. 1 30 (1 992), 41-68; Barbas III, Methods: Companion Methods Enzymol. 2 (1 991), 1 1 9 and Plückthun, Immunochemistry (1 994), Marcel Dekker Inc., Chapter 9, 210-235.
  • equivalent binding ability means an identical binding affinity and / or specificity, i.e. To understand epitope recognition as in the specifically disclosed sequences.
  • the present invention furthermore relates to a vector which contains at least one copy of a nucleic acid according to the invention.
  • This vector can be a prokaryotic vector or a eukaryotic vector.
  • prokaryotic vectors are plasmids, cosmids and bacteriophages. Such vectors are, for example, by Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Eddition (1 989), Cold Spring Harbor Laboratory Press, described in detail in Chapters 1 to 4.
  • a prokaryotic vector is preferably a plasmid or a phage.
  • the vector can also be a eukaryotic vector, e.g. a yeast vector, an insect vector (baculovirus) or a mammalian vector (plasmid vector or viral vector).
  • a eukaryotic vector e.g. a yeast vector, an insect vector (baculovirus) or a mammalian vector (plasmid vector or viral vector). Examples of eukaryotic vectors are described in Sambrook et al., Supra, chapter 1 6 and Winnacker, genes and clones, an introduction to genetic engineering (1 985), VCH publishing company, in particular chapters 5, 8 and 10.
  • Another object of the present invention is a cell which expresses a nucleic acid according to the invention, or a cell which is transformed with a nucleic acid according to the invention or with a vector according to the invention.
  • the cell can be a prokaryotic cell (for example a gram-negative bacterial cell, in particular E. coli) or a eukaryotic cell (for example a yeast, plant or mammalian cell). Examples for Suitable cells and methods for introducing the nucleic acid according to the invention into such cells can be found in the above references.
  • Another object of the present invention is a polypeptide which is encoded by a nucleic acid according to the invention, in particular a recombinant polypeptide.
  • the polypeptide particularly preferably contains the variable domain of the H or / and L chain of a human antibody.
  • a polypeptide which has antibody properties and is composed of a heavy chain or a functional derivative thereof and a light chain or a functional derivative thereof as subunits is particularly preferred.
  • the polypeptide can be composed of two separate chains or can be present as a single chain polypeptide.
  • Yet another object of the present invention is an antibody against a polypeptide according to the invention which is directed against a region of the polypeptide which is responsible for the recognition of the antigen.
  • This antibody can be a polyclonal antiserum, a monoclonal antibody or a fragment of a polyclonal or monoclonal antibody (for example a Fab, F (ab) 2 ,, Fab 'or F (ab') 2 fragment).
  • the antibody is preferably directed against the CDR3 region of the heavy and / or light antibody chain of the polypeptide according to the invention or a region thereof.
  • Antibodies of this type can be obtained by methods known per se by immunizing a test animal with a peptide or polypeptide which contains a CDR3 region according to the invention and obtaining the resulting polyclonal antibodies from the test animal. Furthermore, monoclonal antibodies can be obtained by fusing an antibody-producing B cell of the experimental animal with a leukemia cell according to the Köhler and Milstein method or a further development thereof. In addition, recombinant antibodies which are directed against the CDR3 region of the polypeptide according to the invention can also be obtained by patterning a suitable one Phagemid library, for example a phageimid library from a healthy human donor, can be obtained, a polypeptide according to the invention being used as the antigen.
  • a suitable one Phagemid library for example a phageimid library from a healthy human donor
  • the invention also relates to a pharmaceutical composition which contains a nucleic acid, a vector, a polypeptide, an antibody or a cell as mentioned above as the active component, optionally together with other active components and pharmaceutically customary auxiliaries, additives or carriers.
  • the pharmaceutical composition can be used to produce a diagnostic or therapeutic agent.
  • diagnostic applications are the diagnosis of AlTP or a predisposition to AlTP.
  • Another preferred diagnostic application is monitoring the course of the disease in AlTP.
  • the use of the pharmaceutical composition as a diagnostic agent can include, for example, the detection of a B cell subpopulation which expresses a polypeptide according to the invention as an antibody.
  • the detection of this antibody can, for example, at the nucleic acid level, e.g. by means of a nucleic acid hybridization assay, optionally with preceding amplification.
  • the detection can also be carried out at the protein level by an immunoassay using antigens or antibodies which react specifically with the polypeptide.
  • the pharmaceutical composition according to the invention can also be used in the therapeutic field, in particular for the prevention or therapy of AlTP.
  • This therapeutic application can be based, for example, on stimulating the production of anti-autoantibodies.
  • the autoantibody polypeptide according to the invention can be administered to a patient. thereby causing and / or stimulating the formation of anti-idiotypic antibodies.
  • This administration can be carried out according to customary immunization protocols (Fox et al., J. Pharmacol. Exp. Ther. 279 (1 996), 1 000-1 008; Whittum-Hudson et al., Nat. Med. 2 (1 996), 1 1 1 6-1 1 21; Jardieu, Curr. Opin. Immunol.
  • the expression of antibody genes can be specifically inhibited by administration of suitable antisense nucleic acids.
  • the antiidiotypic antibody polypeptide according to the invention can be administered to a patient in order to achieve a direct inhibition of autoantibody activity.
  • autoantibody polypeptides according to the invention show that they are surprisingly able to inhibit the binding of fibrinogen to platelets.
  • the autoantibody polypeptides and antidiotypic antibody polypeptides according to the invention can therefore optionally be used in combination as agents for modulating blood coagulation, in particular for preventing thrombosis, for example after heart attacks, strokes or in the case of venous thrombosis with pulmonary embolism or ischemia etc.
  • murine monoclonal antibodies fibrinogen antagonists, for example, were the monoclonal antibody 7E3 (see. Eg, US Patent 5,440,020) or fragments thereof (such as the commercially available Fab fragment ReoPro ®) or short synthetic peptides used for therapeutic purposes.
  • Murine monoclonal antibodies and antibody fragments have the disadvantage that they lead to undesirable side reactions in the treatment of human patients due to their immunogenicity, while short peptides are generally broken down very quickly.
  • polypeptides according to the invention have the advantage that they consist of amino acid sequences of human origin and therefore have fewer undesirable side effects than corresponding murine ones Antibodies or antibody fragments, and that their size does not cause them to break down as quickly as peptides.
  • the invention thus relates to the use of a nucleic acid according to the invention, in particular a nucleic acid coding for an autoantibody polyeptide, a vector containing this nucleic acid, a cell transformed with the nucleic acid or the vector, a polypeptide encoded by the nucleic acid or a pharmaceutical composition comprising a or contains several of the substances mentioned, for the preparation of an agent for influencing and in particular inhibiting the binding of fibrinogen to platelets.
  • the agent is preferably used to modulate blood coagulation, in particular for the dissolution of thrombi and / or for the prevention of thrombus formation.
  • the pharmaceutical composition according to the invention can be administered according to protocols already established for murine antibodies or antibody fragments.
  • Yet another object of the invention is a method for obtaining phagemid clones which express nucleic acids which code for autoantibodies against GPIIb / llla or for antiidiotypic antibodies directed against these autoantibodies, characterized in that a phagemid library made up of human lymphocytes Produces donors and wins the desired phagemid clones by affinity selection, including negative and positive selection steps.
  • the method also includes recovering antibody-encoding nucleic acids from the clones and / or using the antibody-encoding nucleic acids to express recombinant antibody chains, derivatives, or fragments thereof.
  • the invention is further illustrated by the following examples, figures and sequence protocols. Show it:
  • SEQ ID No. 1 The nucleotide sequence of the H chain of an antibody according to the invention (phagemid clone PDG7), where
  • CDR complement determining region
  • SEQ ID No. 2 the amino acid sequence to that in SEQ ID No. 1 nucleotide sequence shown, wherein FR1 from A.S. 1 -30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-65, FR3 by A.S. 66-97, CDR3 by A.S. 98-
  • SEQ ID No. 3 shows the nucleotide sequence of the L chain of a polypeptide according to the invention (phagemid clone PDG7), FR1 from bp 1-60, CDR1 from bp 61-99, FR2 from bp 100-
  • SEQ ID No. 4 the amino acid sequence to that in SEQ ID No. 3 indicated nucleotide sequence, FR1 from A.S. 1 -20,
  • SEQ ID No. 5 shows the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone PDG 1 3), FR1 from bp 1-90, CDR1 from bp 91-109, FR2 from bp 1 06-1 47, CDR2 from bp 148-1 98, FR3 from bp 1 99-294, CDR3 from bp 295-336 and FR4 from bp 337-369 are sufficient,
  • SEQ ID No. 6 the amino acid sequence of the in SEQ ID No. 5 nucleotide sequence shown, wherein FR1 from A.S. 1 -30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-66, FR3 by A.S. 67-98, CDR3 by A.S. 99-1 1 2 and FR4 by A.S. 1 1 3-1 23 is enough
  • SEQ ID No. 7 shows the nucleotide sequence of the L chain of a polypeptide according to the invention (phagemid clone PGD1 3), FR1 from bp 1 -60, CDR1 from bp 61 -99, FR2 from bp 1 00-144, CDR2 from bp 145-1 65, FR3 from bp 1 66-261, CDR3 from bp 262-294 and FR4 from bp 295-333 is sufficient,
  • SEQ ID No. 8 the amino acid sequence of the in SEQ ID No. 7 nucleotide sequence shown, wherein FR1 from A.S. 1 -20, CDR1 by A.S. 21-33, FR2 by A.S. 34-48, CDR2 by A.S. 49-55, FR3 by A.S. 56-87, CDR3 by A.S. 88-98 and FR4 by A.S. 99-1 1 1 is enough
  • SEQ ID No. 9 shows the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-X1 6), FR1 from bp 1 -90, CDR1 from bp 91 -105, FR2 from bp 1 06-1 47, CDR2 from bp 148-1 98, FR3 from bp 1 99-288,
  • SEQ ID No. 10 the amino acid sequence of the in SEQ ID No. 9 nucleotide sequence shown, wherein FR1 from A.S. 1 -30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S.
  • SEQ ID No. 1 1 the nucleotide sequence of the L chain of a polypeptide according to the invention (phagemid clone AI-X1 6), where FR1 from bp 1 to 60, CDR1 from bp 61-1 02, FR2 from bp 1 03-1 47, CDR2 from 1 48-1 68, FR3 from bp 1 69-264, CDR3 from 265-291 and FR4 from bp 292-375 are sufficient,
  • SEQ ID No. 1 2 the amino acid sequence of the in SEQ ID No. 1 1 nucleotide sequence shown, wherein FR1 from A.S. 1 -20, CDR1 by A.S. 21-34, FR2 by A.S. 35-49, CDR2 by A.S. 50-56, FR3 by A.S. 57-88, CDR3 by A.S. 89-97 and FR4 by A.S. 89-1 25 is enough
  • SEQ ID No. 1 3 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-X20), FR1 from bp 1-90, CDR1 from bp 91-105, FR2 from bp
  • SEQ ID No. 14 the amino acid sequence of the in SEQ ID No. 1 3 nucleotide sequence shown, wherein FR1 from A.S. 1 -30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-65, FR3 by A.S. 66-97, CDR3 by A.S. 98-1 1 1 and FR4 by A.S. 1 1 2-1 22 is enough
  • SEQ ID No. 1 5 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-X39), FR1 from bp 1 -90, CDR1 from bp 91 -105, FR2 from bp 1 06-147, CDR2 from pb 1 48-1 98, FR3 from bp 1 99-294, CDR3 from bp 295-339 and FR4 from 340-372 ranges, SEQ ID No.
  • SEQ ID No. 17 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-X40), FR1 from bp 1-90, CDR1 from bp 91-105, FR2 from bp 106-147, CDR2 from bp 148-198, FR3 from bp 199 -297.
  • SEQ ID No. 18 the amino acid sequence of the in SEQ ID No. ⁇ nucleotide sequence shown, wherein FR1 from A.S. 1 to 30, CDR1 from A.S.31-35, FR2 from A.S.36-49, CDR2 from
  • SEQ ID No. 19 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-X2), FR1 from bp 1-90, CDR1 from bp 91-105, FR2 from bp 106-147, CDR2 from bp 148-195, FR3 from bp 196-291, CDR3 from bp 292-327 and FR4 from bp 328-360 are sufficient,
  • SEQ ID No.20 the amino acid sequence of the nucleotide sequence shown in SEQ ID No.19, where FR1 from A.S. 1 to 30, CDR1 by A.S.31-35, FR2 by A.S.36-49, CDR2 by A.S.50-65, FR3 by A.S.66-97, CDR3 by A.S.98-109 and FR4 by A.S. 110-120 is enough
  • SEQ ID No.21 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-B14), wherein FR1 by bp 1 -90, CDR1 by bp 91 -1 05, FR2 by bp 106-147, CDR2 by bp 1 48-1 98, FR3 by bp 1 99-294, CDR3 by bp 295-336 and FR4 by bp 337 -369 is enough;
  • Position 69 can be a T, position 76 a K, position 84 an N, position 85 an S and / and position 95 a Y.
  • SEQ ID No. 22 the amino acid sequence of the in SEQ ID No. 21 shown nucleotide sequence, FR1 from A.S. 1 to 30,
  • SEQ ID No. 23 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-B1 8), FR1 from bp 1 -90, CDR1 from bp 91 -105, FR2 from bp 106-1 47, CDR2 from bp 1 48-1 98, FR3 from bp 1 99-294, CDR3 from bp 295-333 and FR4 from bp 334-366 ranges; 0
  • a C can be at position 7 Position 1 3 a G, position 1 6 a C, position 56 an A, position 94 a T, position 97 a G, position 1 55 a T, position 1 73 a C, position 223 a T , a T or a C at position 252, a G at position 261, a G at position 267, a at position 271
  • Position 33 an A, position 52 an I, position 58 an A, position 75 an S, position 84 an S, position 87 an R, position 89 an E, position 91 an T, position 92 an A or / and a V at position 93 may be present.
  • SEQ ID No. 24 the amino acid sequence of the in SEQ ID No. 23 nucleotide sequence shown, wherein FR1 from A.S. 1 to 30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-66, FR3 by A.S. 67-98, CDR3 by A.S. 99-
  • SEQ ID No. 25 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-B24), FR1 from bp 1-90, CDR1 from bp 91-105, FR2 from bp
  • Position 3 a Q position 5 a V, position 1 1 a V, position 1 6 an R, position 23 an A, position 30 an S, position 31 an S, position 33 a G, on Position 34 an M, position 47 a W, position 49 an A, position 50 a V, position 53 a Y, position 54 a D, position 56 an S, position 58 a K, position 79 an L, at position 84 an N, at position 97 an A or / and at position 98 a K.
  • SEQ ID No. 26 the amino acid sequence of the in SEQ ID No. 25 nucleotide sequence shown, wherein FR1 from A.S. 1 to 30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-66, FR3 by A.S. 67-98, CDR3 by A.S. 99-1 10 and FR4 by A.S. 1 1 1 -1 21 is enough
  • SEQ ID No. 27 the nucleotide sequence of the L chain of a polypeptide according to the invention (phagemid clone AI-B24), FR1 from bp 1-60, CDR1 from bp 61-96, FR2 from bp 97-1 38, CDR2 from bp 1 39-1 59, FR3 from bp 1 60-255,
  • CDR3 from bp 256-282 and FR4 from bp 283-366 ranges; The following variations of the nucleotide sequence were also found: A C or a T at position 4, a G at position 37, an A at position 40, a G at position 50, an A at position 67, an A at position 72 Position 1 33 an A, position 1 36 a T, position
  • Position 94 may be an H.
  • SEQ ID No. 28 the amino acid sequence of the sequence shown in SEQ ID No. 27 nucleotide sequence shown, wherein FR1 from A.S. 1 to 20, CDR1 by A.S. 21-32, FR2 by A.S. 33-46, CDR2 by
  • SEQ ID No. 29 the nucleotide sequence of the H chain of a polypeptide according to the invention (phagemid clone AI-B38), where
  • SEQ ID No. 30 the amino acid sequence of the in SEQ ID No. 29 nucleotide sequence shown, wherein FR1 from A.S. 1 to 30, CDR1 by A.S. 31-35, FR2 by A.S. 36-49, CDR2 by A.S. 50-66, FR3 by A.S. 67-98, CDR3 by A.S. 99-
  • Figure 1 shows the inhibition of the binding of autoantibody phabs (PDG-X) to GPIIb / llla by adding the anti-idiotypic antibody Phab AI-X1 7.
  • FIG. 2 shows the inhibition of the binding of autoantibody phabs (PDG-B) to platelets by anti-idiotypic antibody phabs Al-B,
  • Figure 3 shows the binding of autoantibody phabs to untreated
  • FIG. 4 shows the inhibition of fibrinogen binding to GPIIb / llla by autoantibody phabs, Figure 5-7, the inhibition of the binding of autoantibody Phabs to GPIIb / IIIa by the antibody 7E3 and the antibody fragment ReoPro ®.
  • Platelet-enriched plasma was prepared from EDTA-anticoagulated blood samples from healthy human donors by differential centrifugation. The platelets were isolated by centrifugation at 2000 g for 15 minutes, washed six times in citric acid buffer (pH 6.2) with 50 mmol / l sodium citrate, 100 mmol / l NaCl and 125 mmol / l dextrose and finally resuspended in the same buffer .
  • Thrombasthenic platelets were obtained from a 14 year old boy suffering from Glanzmann type I thrombasthenia using the same enrichment protocol.
  • Murine monoclonal antibodies were used which recognize the complex form of GPIIb / llla as well as antibodies which selectively recognize GPIIb or GPIIIa. These antibodies were obtained using conventional immunization protocols using the appropriate antigens and are not associated with AlTP. The production of such antibodies is in Kouns et al. (J. Biol. Chem. 267 (1 992), 1 8844-1 8851), Steiner et al. (Biochim. Biophys. Acta 1 1 1 9 (1 992), 1 2-21) and Häring et al. (Proc. Natl. Acad. Sci. USA 82 (1 985), 4837-4841).
  • a combinatorial Fab library was developed according to the method described by Vogel et al. (Eur. J. Immunol. 24 (1 994), 1 200-1 207) using peripheral blood lymphocytes from a healthy pre-immunized human donor. All enzymes and oligonucleotides were obtained from Boehringer Mannheim GmbH (Mannheim, Germany) with the exception of Taq polymerase (Perkin Elmer, NJ, USA). The primer for the PCR amplification of the H and L chains of the Fab molecules, the VCSM 1 3 helper phage and the Escherichia coli strain XL-Blue were obtained from Stratacyte (La Jolla, CA, USA).
  • the phagemid pComb3 was obtained from the Scripps Research Institute (La Jolla, CA, USA). The cloning, the transformation into XL blue cells and the production of phabs were carried out as described by Barbas III and Lerner, Methods: Companion Methods Enzymol. 2 (1 991), 1 1 9). The phabs were precipitated with 4% (w / v) polyethylene glycol 8000 and 3% (w / v) NaCl and resuspended in PBS pH 7.4. The resulting expression library contains 1 x 10 7 specificities.
  • GPlIb / llla-specific phabs were prepared by a total of 5 rounds of affinity selection ("panning"). After pre-absorption (negative selection) with 5 x 10 7 thrombasthenic platelets, the phabs were incubated with 1 0 8 normal platelets for 45 min (positive selection). Bound phabs were then eluted with 0.05 mol / l sodium citrate pH 2.5 and neutralized with 1 mol / l Tris buffer. After each round of panning, the enrichment of GPIIb / llla specific phabs was followed by titration of the phage colony-forming units. After five rounds of selection, an enrichment of the eluted phabs by a factor of more than 100 was found.
  • the pool of phabs obtained after the fourth round of selection was analyzed in more detail for its GPIIb / llla specificity.
  • 40 Phab clones were selected at random and their binding specificity was determined in an immunodot assay.
  • 1 ul normal and thrombasthenic platelets (10 9 ml) and purified GPIIb / llla (500 ug / ml) were dropped on nitrocellulose strips (Millipore Corporation, Bedford, MA, USA). The strips were blocked in TBS with 0.15% casein (TBS-casein) and then incubated overnight with the phabs diluted in TBS-casein.
  • TBS-0.1% Tween 20 TBS-0.1% Tween 20
  • the bound phabs were washed with 4-chloro-1 - ⁇ - naphthol (Merck, Darmstadt, Germany) after incubation with horseradish peroxidase-conjugated polyclonal rabbit anti-phage antibody (Vogel et al., supra) diluted 1: 1000 in TBS-casein.
  • the binding of phabs to platelets and purified GPIIb / llla was also tested after denaturing the proteins by heating (70 ° C.) or by acid treatment (pH 2 with 0.5 N HCl) before the spotting.
  • the anti-GPIIb / llla phabs were dropped on nitrocellulose.
  • the filters were washed for 4 hours with peroxidase-labeled mouse anti-human-, -K (The Binding Site Limited, Birmingham, England) and -Fd antibodies (from myeloma cell line HP6045, ATCC1757, Rockville, MD, USA ) diluted 1: 1000 incubated in TBS casein and developed with chemiluminescence (ECL, Amersham, Switzerland, Zurich, Switzerland).
  • the epitope specificity of Phabs was determined by an inhibition test using various monoclonal antibodies (see point 4). 1 ⁇ l thawed normal and thrombasthenic platelets (1 0 9 / ml), purified GPIIb / llla (500 ⁇ g / ml), a peptide fragment of GPIIIa (amino acids 468-690, 500 ⁇ g / ml) and the cytoplasmic section of GPIIb / llla (500 ⁇ g / ml) were dripped in duplicate onto nitrocellulose strips. After blocking, the Phab clones (0.4 ug / ml Fab) were incubated overnight with or without monoclonal antibody (1 ug / ml). The bound phabs were detected by peroxidase-labeled anti-PHage antibodies and 4-chloro-1- ⁇ -naphthol.
  • the blocking of the binding of autoantibodies from patients to GPIIb / llla by the anti-GPIIb / llla phabs found was determined by inhibition tests.
  • two of the phabclones identified as described above (PDG 1 6, PDG31) were used.
  • Binding of autoantibodies from 8 AlTP patients was inhibited by anti-GPIIb / llla phabs.
  • the inhibition range was 10 to 46%, 32 to 60% and 20 to 67% for PTG1 6, PTG31 and the pool of the two phabs.
  • the binding of autoantibodies from 4 AlTP patients was not changed by these phabs.
  • Autoantibodies from patients with primary and disease-associated AlTP were in both groups.
  • Plasmid DNA was purified from four Phabklonen the group A and 4 clones of the group with the Nukleobon ® AX purification PC 20 (Macherey-Nagel AG, Oensingen, Switzerland). Nucleic acid sequencing was carried out on an ABI373A sequencing system using a PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit. The primers were obtained from Microsynth, Balgach, Switzerland. The following primers were used for sequencing the H chain: Ch 1 (5'-CGC TGT GCC CCC AGA GGT-3 ') and PCH (5'-GGC CGC AAA TTC TAT TTC AAG G-3').
  • the following primers were used for sequencing the L chain: C ⁇ (5'-GAG ACA CAC CAG TGT GGC-3 '), Ck (5'-CAC AAC AGA GGC AGT TCC-3') and PCL (5'-CTA AAC DAY CTA GTC TCC-3 ').
  • the amino acid sequences derived from the DNA sequence were compared with the GenEMBL library and assigned to parent lines VH and V ⁇ families.
  • VH and V ⁇ nucleotide sequences of the 4 phabclones in each group were analyzed by automated sequencing and with known stem lines -Generic sequences compared (Tables 3 and 4). Within each group there was 100% homology in the deduced amino acid sequences of the H and L chains. In contrast, the homology between groups A and B was only 36.9% for the H chain and 81.9% for the L chain amino acid sequences.
  • group A clones show the highest degree of sequence identity with the stem line gene VH4.1 1 of the V H 4 family (Sanz, et al. EMBO J. 8 (1 989), 3741-3748). There were 7 amino acid differences in the framework region (FR) and 8 in the complement-determining region (CDR). Group B clones differed from the most homologous stem line sequence 1.9111 of the V H 3 family (Berman et al., EMBO J. 7 (1 988), 727-738) by four amino acids in FR and one in CDR.
  • FR framework region
  • CDR complement determining region.
  • the upper sequences (VH4.1 1; 1.9111; DPL2) are given for comparison purposes and represent the deduced amino acid sequence for the most closely related published stem line gene sequence. Dashes 0 indicate identity.
  • M85255 refers to the EMPL / GenBank label number and means the deduced amino acid sequence of the human anti-GPIIb autoantibody 2E7 (Kunicki et al., J. Autoimmun. 4 (1991), 433-446).
  • the first three amino acids are determined by the vector sequence of pComb3.
  • Table 4 shows the assignment of clones of groups A and B to known stem lines V gene sequences according to the amino acid homology
  • sequences for anti-idiotypic antibodies were identified by the phagemid technique.
  • the clone PDG 16 selected in Example 1 was used as the antigen. There was no negative pre-selection.
  • a pool of combinatorial phab libraries the specificities of a nonimmune and a red blood cell immobilized library of peripheral B lymphocytes and a nonimmune library of B lymphocytes from tonsils were used.
  • the pool of phabs obtained after the fourth round of panning was analyzed. For this purpose, 40 Phab clones were selected at random and their binding specificity was determined. 25 of the selected clones reacted with anti-GPIIb / llla phab.
  • DNA sequence analysis of group I phab clones showed complete identity in the sequences coding for the heavy chain except for one amino acid in the CDR2 region and in those coding for the light chain Sequences a complete identity.
  • a comparison with known stem line gene sequences showed approx. 85% homology to the H chain sequence VH3 and approx. 90% homology to the sequence of the L chain family V- ⁇ ll.
  • DNA sequence analysis of the H chain gene was carried out on one representative of each of the Phab clones of groups II, III and IV. The results of this sequence analysis and the comparison with known stem line gene sequences are summarized in Tables 6 and 7a.
  • the result of an inhibition study is shown in FIG. 1.
  • the inhibition of the binding of AI-X1 7 to PDG-A by purified GPIIb / llla was determined by an immunodotassay. 660 and 220 ng PDG-A Phab were applied to nitrocellulose. The antigen was treated for 2 h with GPIIb / llla in concentrations in the range from 50 ⁇ g / ml to 50 ng / ml and with a buffer solution as a control and then for two more hours the phage clone AI-X1 7 (final concentration 10 12 / ml) incubated. The bound phages were detected with peroxidase-conjugated polyclonal rabbit anti-phage antibody and electrochemiluminescence.
  • Phab AI-X17 (Group I) can inhibit the binding of group A autoantibody phabs (PDG-X) to the glycoprotein llb / llla. This means that AI-X1 7 recognizes the antigen binding site on PDG-A.
  • Another clone AI-X2 which binds to PDG-A, was sequenced. Like the clones AI-X20, 39 and 40, this clone has only a heavy chain, but not an easy one.
  • the heavy chain can be alone, optionally as a dimer, with sufficient specificity and affinity for the antigen, i.e. PDG-A, bind.
  • Example 2.1 sequences for further anti-idiotypic antibodies were identified by the phagemid technique.
  • a clone PDG-B selected in Example 1 was used as the antigen.
  • Group 1 (14 clones) reacted only with group B autoantibody phab clones, while group II phab clones (8 clones in total) reacted with both group A and B phab clones.
  • the group III phab clones (a total of 1 2 clones) also reacted with murine monoclonal anti-GPIIb / llla antibodies, with purified serum immunoglobulin (IVIgG) or F (ab ') 2 fragments thereof and with Anti-IgE Fab.
  • group IV did not react with any of the substances mentioned. The results of these specificity studies are summarized in Table 5b.
  • AI-B1 4 and 1 7 are identical.
  • AI-B34 and 40 are also identical to AI-B1 8.
  • the inhibition of PDG-B binding to platelets by Al-B phabs is shown in FIG. 2.
  • a platelet-rich plasma (10 7 platelets in total) was incubated with biotinylated PDG-B in the presence or absence of Al-B phabs and using helper phages as a control.
  • the platelets were fixed with paraformaldehyde and bound PDG-B was detected with R-phycoerythrin (RPE) -labeled streptavidin. 1 0,000 events were counted in a FACScan device and the mean value of fluorescence ( ⁇ SD) was recorded.
  • the strongest inhibition (> 60%) was achieved with the clones AI-B1 8, 24 and 38.
  • the inhibition of binding shows an interaction of Al-B clones with the antigen-binding site on PDG-B.
  • V ⁇ family stem lines Al-X and Al-B) gen (%) * gen (%) * ""
  • AI-B14 V H 3 DP46 91 - - -
  • AI-B38 H 1 DP5 98 - - -
  • FR framework region
  • CDR complement determining region.
  • the upper sequences (DP47, DP49, DP31, DP71 and DPL10) are given for comparison purposes and represent the most closely related known parent line sequence. Dashes indicate identity.
  • the first three amino acids (QVK) are due to the vector sequence of
  • AI-B18 K-LE M -HT— -_T v PR — T-DDSGI EDGTTVPSQPLEF WGQGTRVTVSS
  • AI-B24 K-L L G S N K-AI- Y-S A — SN-G-T V S VR GSGSYLGYYFDY WGQGTLVTVSS
  • FR framework region
  • CDR complement determining region.
  • the upper sequences (DP46, DP10, DP49, DP5 and VL3h) are given for comparison purposes and represent the most closely related known parent line sequence. Dashes indicate identity.
  • the first three amino acids (QVK) are determined by the vector sequence of pComb3.
  • a platelet-rich plasma was incubated with 10 mM EDTA for 30 min. Biotinylated PDG-B and PDG-A polypeptides were added and incubated for 1 h at room temperature. The binding of PDG-A and PDG-B to platelets was measured by flow cytometric analysis using phycoerythrin-labeled streptavidin.
  • Plasma rich in platelets 250 x 10 9 / l was freshly prepared and kept under 5% CO 2 .
  • the plasma was activated by different dilutions of ADP (maximum concentration 410 // M) in the absence or in the presence of PDG-A or PDG-B (maximum amount 10 ⁇ g Fab).
  • the aggregation was measured in a Rodell 300BD-5 aggregometer (Baxter AG, Düdingen, CH).
  • polyclonal anti-Fab antiserum was added to the activated platelets after addition of PDG-A or PDG-B.
  • the bound fibrinogen was with Rat anti-human fibrogen antibody, biotinylated mouse anti-rat antibody and a conjugate of streptavidin and biotinylated horseradish peroxidase (Amersham Pharmacia Biotech Europe GmbH, D Wegdorf, CH) using an ELISA Easy reader (EAR340AT, SLT- Instruments, Austria) measured at 405 nm.
  • Platelet-rich plasma (230 x 10 9 / l) was treated for 1, 5 h with PDG-B or PDG-A (200 or 400 // g / ml) with or without the murine monoclonal antibody 7E3 or its Fab fragment ReoPro ® (total amount of Fab in the range of 10 14 to 10 10 ) incubated.
  • PDG-B and PDG-A to platelets was measured by flow cytometric analysis using phycoerythrin-labeled streptavidin.
  • the tested recombinant anti-GPIIb / llla Fab autoantibody fragments show no binding to platelets which had been pretreated with 10 mM EDTA. This shows that the autoantibody fragments recognize only an antigen that is intact in its conformation (FIG. 3).
  • CAG GTG AAA CTG CTC GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG 48 Gin Val Lys Leu Leu Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 115 120 125
  • MOLECULE TYPE Protein
  • CAG GTG AAA CTG CTC GAG TCT GGG GGA GGC GTG GTC CAC CCT GGG AGG 48 Gin Val Lys Leu Leu Glu Ser Gly Gly Gly Val Val His Pro Gly Arg 125 130 135
  • GCA ATA CAC TGG GTC CGC CAG GCT CCA GGG AAG GGA CTG GAA TAT GTT 144 Ala Ile His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tyr Val 155 160 165 170

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Abstract

L'invention concerne de nouvelles séquences d'acide nucléique qui codent pour des auto-anticorps et des anticorps anti-idiotypes humains contre la protéine membranaire d'agrégation plaquettaire, de nouvelles séquences aminoacides d'anticorps humains et leur utilisation pour le diagnostic et la thérapie de maladies.
EP98934922A 1997-06-06 1998-06-05 Anticorps recombines anti-gpiib/iiia Withdrawn EP0990034A1 (fr)

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EP0751992B1 (fr) 1994-03-08 2005-11-09 Human Genome Sciences, Inc. Facteur 2 de croissance endotheliale vasculaire
US6040157A (en) 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US6608182B1 (en) 1994-03-08 2003-08-19 Human Genome Sciences, Inc. Human vascular endothelial growth factor 2
US7186688B1 (en) 1994-03-08 2007-03-06 Human Genome Sciences, Inc. Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2
US5932540A (en) 1994-03-08 1999-08-03 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7109308B1 (en) 1994-03-08 2006-09-19 Human Genome Sciences, Inc. Antibodies to human vascular endothelial growth factor 2
US7153827B1 (en) 1994-03-08 2006-12-26 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 and methods of use
WO1998055619A1 (fr) * 1997-06-06 1998-12-10 Asat Ag Applied Science & Technology Anticorps recombines anti-gpiib/iiia
US7223724B1 (en) 1999-02-08 2007-05-29 Human Genome Sciences, Inc. Use of vascular endothelial growth factor to treat photoreceptor cells
WO2002011769A1 (fr) 2000-08-04 2002-02-14 Human Genome Sciences, Inc. Facteur 2 de croissance endothéliale (vegf-2)
US20030228309A1 (en) * 2000-11-08 2003-12-11 Theodora Salcedo Antibodies that immunospecifically bind to TRAIL receptors
DE10057443A1 (de) * 2000-11-20 2002-05-23 Asat Ag Applied Science & Tech Rekombinante Anti-GPIIB/IIIA-Antikörper als Mittel zur Hemmung der Angiogenese
FI20010492A0 (fi) * 2001-03-12 2001-03-12 Licentia Oy Uuden von Willebrandin tekijän A3-domeenissa sijaitsevan funktionaalisen sitomiskohdan identifiointi verihiutaleintegriini alfaIIbbeta3 :a varten
EP1385864B1 (fr) * 2001-04-13 2010-06-09 Human Genome Sciences, Inc. Anticorps anti-VEGF-2
US7402312B2 (en) 2001-04-13 2008-07-22 Human Genome Sciences, Inc. Antibodies to vascular endothelial growth factor 2 (VEGF-2)
US20090226429A1 (en) * 2001-05-25 2009-09-10 Human Genome Sciences, Inc. Antibodies That Immunospecifically Bind to TRAIL Receptors
US20050129616A1 (en) * 2001-05-25 2005-06-16 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to TRAIL receptors
US7361341B2 (en) * 2001-05-25 2008-04-22 Human Genome Sciences, Inc. Methods of treating cancer using antibodies that immunospecifically bind to trail receptors
US8455627B2 (en) 2001-10-05 2013-06-04 Affimed Therapeutics, Ag Human antibody specific for activated state of platelet integrin receptor GPIIb/IIIa
ATE364632T1 (de) * 2001-10-05 2007-07-15 Affimed Therapeutics Ag Antikörper menschlichen ursprungs zur hemmung der thrombozytenaggregation
AU2003243651B2 (en) * 2002-06-17 2008-10-16 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Specificity grafting of a murine antibody onto a human framework
CA2515389A1 (fr) 2003-02-28 2004-09-10 Mitsubishi Pharma Corporation Anticorps monoclonal, gene codant pour un tel anticorps, hybridome, composition medicamenteuse et reactif de diagnostic
US7335744B2 (en) * 2003-12-23 2008-02-26 The Regents Of The California University Prostate cancer specific internalizing human antibodies
ES2894398T3 (es) 2011-06-03 2022-02-14 Xoma Technology Ltd Anticuerpos específicos para TGF-beta
US20130084301A1 (en) * 2011-08-30 2013-04-04 Steven Foung Cluster of Neutralizing Antibodies to Hepatitis C Virus
JP7103950B2 (ja) * 2016-04-22 2022-07-20 アクセレロン ファーマ インコーポレーテッド Ａｌｋ７結合性タンパク質及びその使用
CN107056942A (zh) * 2016-10-31 2017-08-18 华东师范大学 抗血小板整合素糖蛋白IIIa单克隆抗体5A10及制备方法和应用
CN111836642A (zh) 2017-10-25 2020-10-27 艾科赛扬制药股份有限公司 Alk7结合蛋白及其用途

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