US20070178082A1 - Stabilized single domain antibodies - Google Patents

Stabilized single domain antibodies Download PDF

Info

Publication number
US20070178082A1
US20070178082A1 US10/534,349 US53434903A US2007178082A1 US 20070178082 A1 US20070178082 A1 US 20070178082A1 US 53434903 A US53434903 A US 53434903A US 2007178082 A1 US2007178082 A1 US 2007178082A1
Authority
US
United States
Prior art keywords
polypeptide construct
single domain
target
domain antibodies
construct according
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.)
Abandoned
Application number
US10/534,349
Inventor
Karen Silence
Marc Lauwereys
Torsten Dreier
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.)
Ablynx NV
Original Assignee
Ablynx NV
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 Ablynx NV filed Critical Ablynx NV
Priority to US10/534,349 priority Critical patent/US20070178082A1/en
Publication of US20070178082A1 publication Critical patent/US20070178082A1/en
Assigned to ABLYNX N.V. reassignment ABLYNX N.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LAUWEREYS, MARC JOZEF, DREIER, TORSTEN, SILENCE, KAREN
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A61K38/166—Streptokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08—Materials for coatings
    • A61L29/085—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • 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
    • 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
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/256—Antibodies, e.g. immunoglobulins, vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/02—Methods for coating medical devices
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/222—Platelet disorders

Definitions

  • Stabilisation of the protein drug can therefore be carried out by choosing an inherently stable protein scaffold and providing methods to bind such scaffold to plasma proteins which occur in high concentrations, such as immunoglobulins or albumin. Binding to plasma protein can be an effective means to improving the pharmacokinetic properties of molecules in general. More precisely, binding to albumin to improve the half-life of proteins has been described: M. S. Dennis et al. ( J. Biol. Chem. 33, 238390, 2002) isolated peptides having affinity for serum albumin.
  • these peptides are inherently unstable and have affinities in the submicromolar range rather than subnanomolar or low nanomolar range, as is the case with conventional antibodies.
  • binding of these peptides to albumin may be sterically hindered.
  • An alternative hybrid molecule with two functional units is based on a heterospecific antibody.
  • Such a hybrid would consist of a bifunctional or heterospecific antibody construct with one entity having specificity and affinity for the target, the second entity having specificity and affinity for a serum protein, such as albumin.
  • a serum protein such as albumin.
  • heterospecific constructs based on conventional antibodies or Fab fragments have several important drawbacks: these are complex, large molecules composed of two polypeptide chains (VH and VL) and therefore difficult and expensive to produce in high amounts in mammalian expression systems.
  • producing bifunctional antibodies composed of 4 chains (2 VH's and 2 VL's) have the inherent risk of resulting in molecules with the unproductive VH-VL combinations and consequent loss of activity.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.
  • polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a polypeptide construct as described above or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is IgE.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least anti-target single domain antibody is anti-IgE VHHs.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is IFN-gamma.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to a sequence represented by SEQ ID NOS: 25 to 27.
  • polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a polypeptide construct as described above directed against a single target wherein said target is involved in a disease process.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic add as described above for use in the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is remains active in the circulation for extended periods of time.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above, or use of a polypeptide construct or nucleic acid as described above, wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a method as described above wherein said anti-serum protein single domain antibodies do not share the same sequence.
  • One embodiment of the present invention is a method as described above wherein said single domain antibodies are Camelidae VHH antibodies.
  • serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.
  • One embodiment of the present invention is a method as described above wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOS: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • One embodiment of the present invention is a composition comprising a polypeptide as described above or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.
  • FIG. 1 phage ELISA to show that HSA-specific nanobodies are present in the library as described in Example 4.
  • FIG. 2 Binding of phages expressing the albumin binders, to plasma blotted on nitrocellulose as described in Example 8.
  • FIG. 3 Coomassie staining of plasma samples on SDS-PAGE as described in example 8.
  • FIG. 4 Binding of purified nanobodies to mouse albumin as determined by ELISA as described in Example 10.
  • FIG. 5 Multiple cloning site of PAX011 for construction of bispecific nanobodies as described in Example 11.
  • FIG. 6 Sandwich ELISA to show the functionality of both nanobodies in the bispecific construct as described in Example 12.
  • FIG. 7 Optimization of ELISA to determine nanobody concentration in 10% plasma or in 10% blood as described in Example 14.
  • FIG. 8 Pharmacokinetics for the monovalent anti-TNF- ⁇ nanobody in mice as determined by ELISA as described in Example 16.
  • FIG. 9 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA as described in Example 16.
  • FIG. 10 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA with K208 as compared to URL49 as described in Example 16.
  • FIG. 11 Pharmacokinetics for the bispecific nanobody MSA24/TNF3E in mice as determined by ELISA as described in Example 16.
  • FIG. 12 Binding to vWF as determined by ELISA, by purified VHH as described in Example 23.
  • FIG. 13 ELISA to test inhibition by VHH of binding of vWF to collagen as described in Example 24.
  • FIG. 14 Sandwich ELISA showing the functionality of both VHHs in a bispecific construct as described in example 27.
  • Table 2 Results after one and two rounds of panning on mouse serum albumin as described in example 5.
  • the present invention relates to a heterospecific polypeptide construct comprising one or more single domain antibodies each directed against a serum protein(s) of a subject, and one or more single domain antibodies each directed against a target molecule(s) and the finding that the construct has a significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target single domain antibody when not part of such a construct.
  • Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678 for example.
  • variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.
  • the one or more single domain antibodies of the polypeptide construct which are directed against a target may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-target single domain antibodies which do not all share the same sequence, but which are directed against the same target, or fragment thereof, one or more antigens thereof.
  • the one or more single domain antibodies of the polypeptide construct which are directed against a serum protein may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-serum protein single domain antibodies which do not all share the same sequence, but which are directed against serum protein, or fragment thereof, one or more antigens thereof.
  • one or more anti-target single domain antibodies of the polypeptide construct may be directed to more than one target (e.g. vWF and collagen).
  • the anti-serum protein single domain antibodies of the polypeptide construct may be directed against more than one serum protein (e.g. serum albumin and fibrinogen).
  • VHHs are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelids as described in WO9404678 (and referred to hereinafter as VHH domains or nanobodies).
  • VHH molecules are about 10 ⁇ smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs.
  • antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Camelids (WO 9749805).
  • anti-albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein.
  • serum albumin which is known to be a carrier protein.
  • some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into ‘unusual’ or non-conventional epitopes such as cavities (WO9749805), the affinity of such VHH's to circulating albumin may be increased.
  • the present invention also relates to the finding that a heterospecific polypeptide construct comprising one or more VHHs directed against one or more serum proteins of a subject, and one or more VHHs directed against one or more target molecule of said subject surprisingly has significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target VHH when not part of said construct. Furthermore, such prolonged half-life is in the range of several days due to the high affinity anti-serum albumin VHH's compared to several hours when using low affinity peptides specific for albumin (Dennis et al, JBC, 277, 35035).
  • Example 16 The extension of the half-life is demonstrated by the inventors herein, for example, in Example 16, and by the polypeptide represented by SEQ ID NO: 5. Furthermore, the said construct was found to exhibit the same favourable properties of VHHs such as high stability remaining intact in mice for at least 19 days (Example 16), extreme pH resistance, high temperature stability and high target affinity.
  • a target according to the invention is any biological substance capable of binding to a heterospecific polypeptide construct of the invention.
  • Targets may be, for example, proteins, peptides, nucleic acids, oligonucleic acids, saccharides, polysaccharides, glycoproteins. Examples include, but are not limited to therapeutic targets, diagnostic targets, receptors, receptor ligands, viral coat proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins, or a fragment thereof.
  • Targets may be native protein or a fragment thereof, a homologous sequence thereof, a functional portion thereof, or a functional portion of an homologous sequence.
  • single domain antibodies in particular VHHs
  • VHHs very low-density antibodies
  • Traditional antibodies are not stable at room temperature, and have to be refrigerated for preparation and storage, requiring necessary refrigerated laboratory equipment, storage and transport, which contribute towards time and expense. Refrigeration is sometimes not feasible in developing countries.
  • manufacture or small-scale production of said antibodies is expensive because the mammalian cellular systems necessary for the expression of intact and active antibodies require high levels of support in terms of time and equipment, and yields are very low.
  • traditional antibodies have a binding activity which depends upon pH, and hence are unsuitable for use in environments outside the usual physiological pH range such as, for example, in treating gastric bleeding, gastric surgery. Furthermore, traditional antibodies are unstable at low or high pH and hence are not suitable for oral administration.
  • VHHs resist harsh conditions, such as extreme pH, denaturing reagents and high temperatures (Ewert S et al, Biochemistry 2002 Mar 19;41 (11):3628-36), so making them suitable for delivery by oral administration.
  • traditional antibodies have a binding activity which depends upon temperature, and hence are unsuitable for use in assays or kits performed at temperatures outside biologically active-temperature ranges (e.g. 37 ⁇ 20° C.).
  • VHHs are more soluble, meaning they may be stored and/or administered in higher concentrations compared with conventional antibodies.
  • the polypeptides of the present invention also retain binding activity at a pH and temperature outside those of usual physiological ranges, which means they may be useful in situations of extreme pH and temperature which require a modulation of platelet-mediated aggregation, such as in gastric surgery, control of gastric bleeding, assays performed at room temperature etc.
  • the polypeptides of the present invention also exhibit a prolonged stability at extremes of pH, meaning they would be suitable for delivery by oral administration.
  • the polypeptides of the present invention may be cost-effectively produced through fermentation in convenient recombinant host organisms such as Escherichia coli and yeast; unlike conventional antibodies which also require expensive mammalian cell culture facilities, achievable levels of expression are high.
  • Examples of yields of the polypeptides of the present invention are 1 to 10 mg/ml ( E. coli ) and up to 1 g/l (yeast).
  • the polypeptides of the present invention also exhibit high binding affinity for a broad range of different antigen types, and ability to bind to epitopes not recognised by conventional antibodies; for example they display long CDR-based loop structures with the potential to penetrate into cavities and exhibit enzyme function inhibition.
  • polypeptides derived from CDR3 could be used therapeutically (Desmyter et al, J Biol Chem, 2001, 276: 26285-90).
  • the polypeptides of the invention are also able to retain full binding capacity as fusion protein with an enzyme or toxin.
  • the present invention also relates to a heterospecific polypeptide construct comprising one or more VHHs each directed against one or more serum proteins of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to the traditional class of Camelidae single domain heavy chain antibodies.
  • the present invention also relates to a heterospecific polypeptide construct comprising one or more VHH each directed against one or more serums protein of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to a class of Camelidae single domain heavy chain antibodies that have human-like sequences.
  • a VHH sequence represented by SEQ ID NO: 12 which binds to TNF-alpha and a second VHH which binds to mouse albumin belongs to this class of VHH peptides.
  • peptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to patients directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.
  • a human-like class of Camelidae single domain antibodies represented by SEQ ID No. 1, 3 and 4 have been described in WO03035694 and contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies.
  • peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.
  • one aspect of the present invention allows for the direct administration of an anti-serum albumin polypeptide, wherein the single domain antibodies belong to the humanized class of VHH, and comprise a sequence represented by any of SEQ ID NO: 1, 3 or 4 to a patient in need of the same.
  • a subject as used herein is any mammal having a circulatory system in which the fluid therein comprises serum proteins.
  • circulatory system include blood and lymphatic systems.
  • animals include, but are not limited to, rabbits, humans, goats, mice, rats, cows, calves, camels, llamas, monkeys, donkeys, guinea pigs, chickens, sheep, dogs, cats, horses etc.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target, and at least one single domain antibodies each directed against one or more serum proteins or polypeptides.
  • the anti-target single domain antibodies may have the same sequence.
  • at least two anti-target single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same target, fragments thereof, or antigen thereof.
  • the anti-serum protein single domain antibodies may have the same sequence.
  • at least two anti-serum protein single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same serum protein, fragments thereof, or antigen thereof.
  • each anti-target single domain antibody may be directed to a different target (e.g. one to vWF and one to collagen).
  • each anti-serum single domain antibody may be directed to a different serum protein (e.g. one to serum albumin and one to fibrinogen).
  • One embodiment of the invention is a heterospecific polypeptide, wherein an anti-serum protein single domain antibody corresponds to a sequence represented by any of SEQ ID NOS:1 to 4 and 28 to 40.
  • the constructs disclosed herein retain the advantageous properties of single domain antibodies (e.g. VHHs) and have a prolonged lifetime in the circulation of an individual. Thus, such constructs are able to circulate in the subject's serum for several days, reducing the frequency of treatment, the inconvenience to the subject and resulting in a decreased cost of treatment. Furthermore, it is an aspect of the invention that the half-life of the heterospecific polypeptide constructs may be controlled by the number of anti-serum protein single domain antibodies present in the construct. A controllable half-life is desirable in several circumstances, for example, in the application of a timed dose of a therapeutic heterospecific polypeptide construct, or to obtain a desired therapeutic effect.
  • single domain antibodies e.g. VHHs
  • a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct.
  • a heterospecific polypeptide construct may be a functional portion of a full-length heterospecific polypeptide construct.
  • a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct.
  • a heterospecific polypeptide construct may be a functional portion of a homologous sequence of a full-length heterospecific polypeptide construct.
  • a heterospecific polypeptide construct may comprise a sequence of a heterospecific polypeptide construct.
  • a single domain antibody used to form a heterospecific polypeptide construct may be a complete single domain antibody (e.g. a VHH) or a homologous sequence thereof.
  • a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a complete single domain antibody.
  • a single domain antibody used to form the heterospecific polypeptide construct may be a homologous sequence of a complete single domain antibody.
  • a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a homologous sequence of a complete single domain antibody.
  • a heterospecific polypeptide construct may be an homologous sequence of the parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a homologous sequence of the parent sequence.
  • an homologous sequence of the present invention may comprise additions, deletions or substitutions of one or more amino acids, which do not substantially alter the functional characteristics of the polypeptides of the invention.
  • the number of amino acid deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • Humanised is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent.
  • Humanising a single domain antibody comprises a step of replacing one or more of amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanisation does not significantly affect the antigen binding capacity of the resulting polypeptide.
  • a humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of any of the following residues either alone or in combination: some VHH contain typical Camelidae hallmark residues at position 37, 44, 45 and 47 with hydrophilic characteristics.
  • VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanisation. Therefore, one aspect of the present invention allows for the formation of a heterospecific polypeptide construct without humanisation of the VHH, when said VHH exhibit high homology to human VH framework regions.
  • a homologous sequence of the present invention may be a sequence of the invention derived from another species such as, for example, camel, llama, dromedary, alpaca, guanaco etc.
  • homologous sequence indicates sequence identity, it means a sequence which presents a high sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with a single domain antibody of the invention, and is preferably characterised by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.
  • a homologous sequence according to the present invention may refer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000 nucleotides able to hybridise to the reverse-complement of the nucleotide sequence capable of encoding a native sequence under stringent hybridisation conditions (such as the ones described by SAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York).
  • a functional portion refers to a single domain antibody of sufficient length such that the interaction of interest is maintained with affinity of 1 ⁇ 10 ⁇ 6 M or better.
  • a functional portion of a single domain antibody of the invention comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target or serum protein.
  • a functional portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.
  • a portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.
  • Targets as mentioned herein such as TNF-alpha, IFN-gamma receptor, serum proteins (e.g. serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, fibrinogen) and IFN-gamma may be fragments of said targets.
  • a target is also a fragment of said target, capable of eliciting an immune response.
  • a target is also a fragment of said target, capable of binding to a single domain antibody raised against the full length target.
  • a fragment as used herein refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.
  • a fragment is of sufficient length such that the interaction of interest is maintained with affinity of 1 ⁇ 10 ⁇ 6 M or better.
  • a fragment as used herein also refers to optional insertions, deletions and substitutions of one or more amino acids which do not substantially alter the ability of the target to bind to a single domain antibody raised against the wild-type target.
  • the number of amino acid insertions deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • the serum protein may be any suitable protein found in the serum of subject, or fragment thereof.
  • the serum protein is serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen.
  • the VHH-partner can be directed to one of the above serum proteins.
  • a single domain antibody directed against a target means single domain antibody that it is capable of binding to its target with an affinity of better than 10 ⁇ 6 M.
  • heterospecific polypeptide constructs disclosed herein may be made by the skilled artisan according to methods known in the art or any future method.
  • VHHs may be obtained using methods known in the art such as by immunising a camel and obtaining hybridomas therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by using phage display.
  • the anti-serum protein single domain antibody may be directed against a polypeptide of a serum protein or a whole protein.
  • the anti-target single domain antibody may be directed against a polypeptide of said target of the whole target. Methods for scanning a protein for immunogenic polypeptides are well known in the art.
  • the single domain antibodies may be joined using methods known in the art or any future method. For example, they may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the single domain antibody may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more anti-target single domain antibodies and one or more anti-serum protein single domain antibodies.
  • a method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO 96/34103.
  • One way of joining multiple single domain antibodies is via the genetic route by linking single domain antibody coding sequences either directly or via a peptide linker.
  • the C-terminal end of the first single domain antibody may be linked to the N-terminal end of the next single domain antibody.
  • This linking mode can be extended in order to link additional single domain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.
  • An aspect of the present invention is the administration of heterospecific polypeptide constructs according to the invention which avoids the need for injection.
  • Conventional antibody-based therapeutics have significant potential as drugs because they have extraordinarily specificity to their target and a low inherent toxicity, however, they have one important drawback: these are complex, large molecules and therefore relatively unstable, and they are sensitive to breakdown by proteases.
  • This means that conventional antibody drugs cannot be administered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation because they are not resistant to the low pH at these sites, the action of proteases at these sites and in the blood and/or because of their large size. They have to be administered by injection (intravenously, subcutaneously, etc.) to overcome some of these problems.
  • heterospecific polypeptides constructs of the present invention overcomes these problems of the prior art, by providing the heterospecific polypeptides constructs of the present invention. Said constructs are sufficiently small, resistant and stable to be delivered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation substantial without loss of activity.
  • the heterospecific polypeptides constructs of the present invention avoid the need for injections, are not only cost/time savings, but are also more convenient and more comfortable for the subject.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.
  • formulation technology may be applied to release a maximum amount of VHHs in the right location (in the stomach, in the colon, etc.). This method of delivery is important for treating, prevent and/or alleviate the symptoms of disorder whose targets that are located in the gut system.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder susceptible to modulation by a therapeutic compound that is able pass through the gastric environment without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the gut system without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract.
  • a formulation according to the invention comprises a heterospecific polypeptide construct as disclosed herein comprising one or more VHHs directed against one or more targets in the form of a gel, cream, suppository, film, or in the form of a sponge or as a vaginal ring that slowly releases the active ingredient over time (such formulations are described in EP 707473, EP 684814, U.S. Pat. No. 5,629,001).
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound to the vaginal and/or rectal tract, by vaginally and/or rectally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the vaginal and/or rectal tract without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target comprising at least one single domain antibody directed against a target, for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung.
  • a formulation according to the invention comprises a heterospecific polypeptide construct as disclosed herein directed against one or more targets in the form of a nasal spray (e.g. an aerosol) or inhaler. Since the construct is small, it can reach its target much more effectively than therapeutic IgG molecules.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound delivered to the upper respiratory tract and lung, by administering to a subject a heterospecific polypeptide construct as disclosed herein wherein one or more single domain antibodies are specific for an antigen related to the disorder, by inhalation through the mouth or nose.
  • VHH compositions in particular dry powder dispersible VHH compositions, such as those described in U.S. Pat. No. 6,514,496.
  • These dry powder compositions comprise a plurality of discrete dry particles with an average particle size in the range of 0.4-10 mm.
  • Such powders are capable of being readily dispersed in an inhalation device.
  • VHH's are particularly suited for such composition as lyophilized material can be readily dissolved (in the lung subsequent to being inhaled) due to its high solubilisation capacity (Muyldermans, S., Reviews in Molecular Biotechnology, 74, 277-303, (2001)).
  • such lyophilized VHH formulations can be reconstituted with a diluent to generate a stable reconstituted formulation suitable for subcutaneous administration.
  • anti-IgE antibody formulations (Example 1; U.S. Pat. No. 6,267,958, EP 841946) have been prepared which are useful for treating allergic asthma.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and lung, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and/or lung without being inactivated, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • One embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa. Because of their small size, a heterospecific polypeptide construct as disclosed herein can pass through the intestinal mucosa and reach the bloodstream more efficiently in subjects suffering from disorders which cause an increase in the permeability of the intestinal mucosa.
  • VHH is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream.
  • this “carrier” is a second VHH which is fused to the therapeutic VHH.
  • Such fusion constructs are made using methods known in the art.
  • the “carrier” VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the intestinal mucosa without being inactivated, by administering orally to a subject a heterospecific polypeptide construct of the invention.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the tissues beneath the tongue effectively.
  • a formulation of said polypeptide construct as disclosed herein, for example, a tablet, spray, drop is placed under the tongue and adsorbed through the mucus membranes into the capillary network under the tongue.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound that is able pass through the tissues beneath the tongue effectively, by sublingually administering to a subject a VHH specific for an antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able to pass through the tissues beneath the tongue.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the tissues beneath the tongue without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct as disclosed herein as a topical ophthalmic composition for the treatment of ocular disorder, such as allergic disorders, which method comprises the topical administration of an ophthalmic composition comprising polypeptide construct as disclosed herein, said construct comprising one or more anti-IgE VHH (Example 1, Example 2).
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the skin without being inactivated, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • a heterospecific polypeptide construct further comprises a carrier single domain antibody (e.g. VHH) which acts as an active transport carrier for transport said heterospecific polypeptide construct, the lung lumen to the blood.
  • a carrier single domain antibody e.g. VHH
  • a polypeptide construct further comprising a carrier binds specifically to a receptor present on the mucosal surface (bronchial epithelial cells) resulting in the active transport of the polypeptide from the lung lumen to the blood.
  • the carrier single domain antibody may be fused to the polypeptide construct. Such fusion constructs made using methods known in the art and are describe herein.
  • the “carrier” single domain antibody binds specifically to a receptor on the mucosal surface which induces an active transfer through the surface.
  • Another aspect of the present invention is a method to determine which single domain antibodies (e.g. VHHs) are actively transported into the bloodstream upon nasal administration.
  • a na ⁇ ve or immune VHH phage library can be administered nasally, and after different time points after administration, blood or organs can be isolated to rescue phages that have been actively transported to the bloodstream.
  • a non-limiting example of a receptor for active transport from the lung lumen to the bloodstream is the Fc receptor N (FcRn).
  • FcRn Fc receptor N
  • One aspect of the invention includes the VHH molecules identified by the method. Such VHH can then be used as a carrier VHH for the delivery of a therapeutic VHH to the corresponding target in the bloodstream upon nasal administration.
  • One embodiment of the present invention is a heterospecific polypeptide construct for use in treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound intraveneously.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound via the bloodstream.
  • the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time.
  • An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time.
  • Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound that is able to circulate in the patients serum for several days, by administering a heterospecific polypeptide construct as disclosed herein to an individual.
  • the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder relating to allergies.
  • An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies.
  • Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies, by administering a heterospecific polypeptide construct as disclosed herein to an individual.
  • the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • an anti-serum single domain antibody of the aforementioned heterospecific polypeptide constructs corresponds to a sequence represented by SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.
  • heterospecific polypeptide construct of the invention corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.
  • Said sequences comprise an anti-TNF-alpha Camelidae VHH.
  • heterospecific polypeptide constructs of the invention corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21 a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.
  • Said sequences comprise an anti-vWF Camelidae VHH.
  • heterospecific polypeptide construct according to the invention corresponds to a sequence represented by any of SEQ ID NOS:25 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.
  • Said sequences comprise an anti-Interferon-gamma Camelidae VHH.
  • Tumor necrosis factor alpha is believed to play an important role in various diseases, for example in inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNF-alpha and the receptors (CD120a, CD120b) have been studied in great detail. TNF-alpha in its bioactive form is a trimer and the groove formed by neighboring subunits is important for the cytokine-receptor interaction. Several strategies to antagonize the action of the cytokine have been developed and are currently used to treat various disease states.
  • a TNF inhibitor which has sufficient specificity and selectivity to TNF may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory diseases.
  • NCE chemical entitly
  • Antibody-based therapeutics on the other hand have significant potential as drugs because they have extraordinarily specificity to their target and a low inherent toxicity.
  • the development time can be reduced considerably when compared to the development of new chemical entities (NCE's).
  • conventional antibodies are difficult to elicit against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with TNF-alpha.
  • heterospecific polypeptide constructs of the present invention wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties such as stability, size, and reliable expressioin. Furthermore, the inventors have found that, despite presence of a groove in multimeric TNF-alpha, the heterospecific polypeptide constructs are still able to achieve strong binding to TNF-alpha
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules.
  • An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules.
  • Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules, by administering a heterospecific polypeptide construct as disclosed herein to an individual.
  • an anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • a target against which a single domain antibody of a heterospecific polypeptide construct is directed is tumor necrosis factor alpha (TNF-alpha).
  • TNF-alpha is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • Anti-target single domain antibodies may be directed against whole TNF-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibody fused to one or more anti-serum protein single domain antibody, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 5 to 18.
  • the anti-TNF-alpha single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to TNF-alpha.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of inflammatory disorders.
  • TNF-alpha is involved in inflammatory processes, and the blocking of TNF-alpha action can have an anti-inflammatory effect, which is highly desirable in certain disorder states such as, for example, Crohn's disease.
  • Oral delivery of these heterospecific polypeptide construct results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain TNF-alpha producing cells.
  • heterospecific polypeptide constructs can neutralise the TNF-alpha locally, avoiding distribution throughout the whole body and thus limiting negative side-effects.
  • Genetically modified microorganisms such as Micrococcus lactis are able to secrete antibody fragments. Such modified microorganisms can be used as vehicles for local production and delivery of antibody fragments in the intestine. By using a strain which produces a TNF-alpha-neutralising heterospecific polypeptide construct, inflammatory bowel disorder could be treated.
  • heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • the anti-TNF-alpha single domain antibodies of the present invention may be derived from VHHs of any class.
  • they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH.
  • VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • SEQ ID NOS: 5 to 18 comprise anti-TNF alpha Camelidae VHH and anti-mouse serum albumin Camelidae VHH.
  • heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (anti-serum protein Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • heterospecific polypeptide construct comprising a sequence corresponding to any of SEQ ID NOS: 5 to 18 surprisingly exhibits higher than expected affinity towards its target and prolonged half-life in the circulatory system.
  • Platelet-mediated aggregation is the process wherein von Willebrand Factor (vWF)-bound collagen adheres to platelets and/or platelet receptors (examples of both are gpla/lla, gplb, or collagen), ultimately resulting in platelet activation. Platelet activation leads to fibrinogen binding, and finally to platelet aggregation.
  • vWF von Willebrand Factor
  • the ability to disrupt platelet-mediated aggregation has many applications including the treatment of disease as mentioned below. Since the heterospecific polypeptide constructs of the invention effective prevent clotting, and the half-life thereof is controllable, they may be used for surgical procedures, for example, which require an inhibition of platelet-mediated aggregation for a limited time period.
  • Monovalent single domain antibodies such as VHHs show surprisingly high platelet aggregation inhibition in experiments to measure platelet aggregation inhibition under high shear: 50% inhibition of platelet aggregation was obtained at a concentration between 4 and 25 nM.
  • the Fab fragment derived from a vWF-specific antibody inhibiting the interaction with collagen, 82D6A3 inhibits 50% of platelet aggregation at approximately a twenty-fold higher concentration (Vanhoorelbeke K. et al, Journal of Biological Chemistry, 2003, 278: 37815-37821).
  • IgG antibodies is not suited to interaction with macromolecules which are starting, or are in the process of aggregating, such as those involved in platelet-mediated aggregation.
  • vWF makes multimers of up to 60 monomers (final multimers of up to 20 million dalton in size). Indeed, it has been shown that not all A3 domains are accessible to 82D6A3 (Dongmei WU, Blood, 2002, 99, 3623 to 3628). Furthermore the large size of conventional antibodies, would restrict tissue penetration, for example, during platelet-mediated aggregation at the site of a damaged vessel wall.
  • VHH molecules derived from Camelidae antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense tissues and for accessing the limited space between macromolecules participating in or starting the process of platelet mediated aggregation.
  • heterospecific polypeptide constructs which modulate processes which comprise platelet-mediated aggregation such as, for example, vWF-collagen binding, vWF-platelet receptor adhesion, collagen-platelet receptor adhesion, platelet activation, fibrinogen binding and/or platelet aggregation.
  • Said heterospecific polypeptide constructs are derived from single domain antibodies directed towards vWF, vWF A1 or A3 domains, gplb or collagen.
  • Anti-target single domain antibodies may be directed against whole vWF, vWF A1 or A3 domains, gplb or collagen or a fragment thereof, or a fragment of a homologous sequence thereof.
  • a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is von Willebrand factor (vWF).
  • vWF von Willebrand factor
  • the target is vWF A1 or A3 domains.
  • the target is gplb.
  • the target is gpla/llA.
  • the target is collagen.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies fused to one or more anti-serum protein VHHs, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 19 to 21.
  • the anti-vWF single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to vWF.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen for use in treating, preventing and/or alleviating the symptoms of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • Said disorders include transient cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis.
  • Said conditions include those arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.
  • One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders or conditions relating to relating to platelet-mediated aggregation or dysfunction thereof, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • the anti-vWF, anti-vWF A1 or anti-vWF A3 or anti-collagen VHHs of the present invention may be derived from VHHs of any class.
  • they may be derived from the class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH.
  • These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • SEQ ID NOS: 19 to 21 comprise anti-vWF VHH and anti-mouse serum albumin VHH.
  • heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen and wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • One aspect of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies, said heterospecific polypeptide construct preventing the interaction of IgEs with their receptor(s) on mast cells and basophils. As such they prevent the initiation of the immunological cascade, an allergic reaction.
  • a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is IgE.
  • Said antibodies may be directed against whole IgE or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 22 to 24.
  • the anti-IgE single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IgE.
  • Anti-target single domain antibodies may be directed against whole IgE-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibody fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of disorders relating to allergies.
  • Said disorders comprise a wide range of IgE-mediated diseases such as hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.
  • One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said VHH is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to allergies, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.
  • Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.
  • the anti-IgE single domain antibodies of the present invention may be derived from VHHs of any class.
  • they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH.
  • Said VHHs may be derived from Camelidae .
  • These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the heterospecific polypeptides correspond to a sequence represented by any of SEQ ID NOS: 22 to 24 , a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • SEQ ID NOS: 22 to 24 comprise anti-IgE Camelidae VHH and anti-mouse serum albumin Camelidae VHH.
  • heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said ant-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (ant-protein serum Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • a heterospecific polypeptide construct as disclosed herein prevents thus reduces or prevents an allergic response due to common or unusual allergens. Furthermore, the construct has a prolonged lifetime in the blood so increasing the therapeutic window.
  • Interferon gamma is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, multiple sclerosis and hyperimmune reactions in the eye. IFN-gamma has also been shown to play a significant role in the pathology of autoimmune diseases. For example, the presence of IFN-gamma has been implicated in rheumatoid arthritis (Brennan et al, Brit. J. Rheum., 31, 293-8 (1992)). Several strategies to antagonize the action of these cytokines have been developed and are currently used to treat various disease states.
  • IFN-gamma in its bioactive form is a dimer and the groove formed by the two subunits is important for its biological activity through interaction with the IFN-gamma receptor.
  • An IFN-gamma inhibitor which has sufficient specificity and selectivity to IFN-gamma may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory disorders.
  • Diseases associated with IFN-gamma include multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, and psoriatic arthritis (U.S. Pat. No. 6,333,032 Advanced Biotherapy Concepts, Inc.).
  • Other diseases include Crohn's disease and psoriasis (U.S. Pat. No. 6,329,511 Protein Design Labs).
  • Yet other diseases are bowel disease, ulcerative colitis and Crohn's disease (EP0695189 Genentech).
  • heterospecific polypeptide constructs of the present invention wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties thereof such as stability, size, and reliable expression. Furthermore, the inventors have found that, despite presence of a groove in multimeric IFN-gamma, the heterospecific polypeptide constructs are still able to achieve strong binding to IF NA-gamma.
  • a target against which one or more anti-target single domain antibodies of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is interferon-gamma (IFN-gamma).
  • IFN-gamma is secreted by some T cells.
  • IFN-gamma stimulates natural killer (NK) cells and T helper 1 (Th1 ) cells, and activates macrophages and stimulates the expression of MHC molecules on the surface of cells.
  • NK natural killer
  • Th1 T helper 1
  • IFN-gamma generally serves to enhance many aspects of immune function, and is a candidate for treatment of disorders where the immune system is over-active e.g. Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-lFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 25 to 27.
  • the anti-IFN-gamma single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IFN-gamma.
  • Anti-target single domain antibodies may be directed against whole IFN-gamma or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of the disorders wherein the immune system is overactive, as mentioned above.
  • Current therapy consists of intravenous administration of anti-IFN-gamma antibodies.
  • Oral delivery of these heterospecific polypeptide constructs results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain IFN-gamma producing cells.
  • These heterospecific polypeptide constructs can neutralise the IFN-gamma locally, avoiding distribution throughout the whole body and thus limiting negative side-effects.
  • heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is over active, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies joined to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive.
  • the anti-IFN-gamma single domain antibodies of the present invention may be derived from VHHs of any class.
  • they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH.
  • These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein VHHs wherein said anti-serum protein VHHs correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • One embodiment of the present invention is a recombinant done comprising nucleic acid encoding a heterospeclfic polypeptide construct according to the invention.
  • said nucleic acid encodes one or more single domain antibodies each directed to a therapeutic or diagnostic target antigen and one or more single domain antibodies directed to a serum protein, said single domain antibodies linked without intervening linkers, or with one or more peptide linker sequences.
  • a linker sequence is any suitable linker sequence known in the art.
  • a linker sequence is a naturally occurring sequence.
  • linkers sequences are that they are not immunogenic or not significantly immunogenic, they can provide sufficient flexibility to the heterospecific polypeptide construct, and are resistant to proteolytic degradation.
  • An example of a linker according to the invention is that disclosed in PCT/EP96/01725 which is derived from the hinge region of VHH.
  • a done comprises nucleic acid encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion, and nucleic acid encoding one or more anti-target single domain antibodies, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • a clone comprises nucleic acid capable of encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS:5 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • Treating, preventing and/or alleviating the symptoms of one or more of the disorders mentioned herein generally involves administering to a subject a “therapeutically effective amount” of heterospecific polypeptide construct.
  • therapeutically effective amount means the amount needed to achieve the desired result or results.
  • an “effective amount” can vary for the various compounds that inhibit a disorder pathway used in the invention.
  • One skilled in the art can readily assess the potency of the compound.
  • the term “compound” refers to a heterospecific polypeptide construct as disclosed herein, a polypeptide represented by SEQ ID NOS: 5 to 27, a homologous sequence thereof, or a homologue thereof, or a nucleic acid capable of encoding said polypeptide.
  • One aspect of the present invention is the use of compounds of the invention for treating or preventing a condition relating to a disorder as mentioned herein (e.g. allergy and/or inflammation), in a subject and comprising administering a pharmaceutically effective amount of a compound in combination with another, such as, for example, aspirin.
  • a condition relating to a disorder as mentioned herein e.g. allergy and/or inflammation
  • another such as, for example, aspirin.
  • the present invention is not limited to the administration of formulations comprising a single compound of the invention. It is within the scope of the invention to provide combination treatments wherein a formulation is administered to a patient in need thereof that comprises more than one compound of the invention.
  • the method would result in at least a 10% reduction in an indicator of the disorder, including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably by 90%.
  • an inhibition of an allergic pathway by inhibition of IgE by a peptide of the invention might result in a 10% reduction in food-specific IgE levels.
  • the compound useful in the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or any animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intranasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes.
  • a mammalian host such as a human patient or any animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intranasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes.
  • the compound of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety.
  • gene therapy methods of delivery See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety.
  • primary cells transfected with the gene for the compound of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells.
  • the present compound may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compound may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the present compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositons which can be used to deliver the compound to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • Useful dosages of the compound can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • the concentration of the compound(s) in a liquid composition will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
  • concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the compound varies depending on the target cell, tumor, tissue, graft, or organ.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • HSA human serum albumin
  • PBLs Peripheral blood lymphocytes
  • cDNA was prepared on 100 ⁇ g total RNA with MMLV Reverse Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides.
  • the cDNA was purified with a phenol/chloroform extraction, followed by an ethanol precipitation and subsequently used as template to amplify the VHH repertoire.
  • the PCR products were digested with Sfil (introduced in the FR1 primer) and BstEll (naturally occurring in FR4). Following gel electrophoresis, the DNA fragment of approximately 400 basepairs were purified from gel and ligated into the corresponding restriction sites of phagemid pAX004 to obtain a library of cloned VHHs after electroporation of Escherichia coli TG1. The size of the library was 1.4 ⁇ 10 7 cfu, and all clones contained insert of the correct size.
  • Eluted phages were allowed to infect exponentially growing E.Coli TG1 cells, and were then plated on LB agar plates containing 100 ⁇ g/ml ampicillin and 2% glucose. A second round was performed with the same conditions as described above. Results are summarized in Table 2.
  • HSA human serum albumin
  • MSA mouse serum albumin
  • a microtiter plate was coated with 5 ⁇ g/ml HSA, with 5 ⁇ /ml mouse serum albumin (MSA) or with PBS-1% casein, overnight at 4° C. Plates were blocked for two hours at room temperature with 300 ⁇ l 1% casein in PBS. The plates were washed three times with PBS-Tween. Periplasmic fraction was prepared for 23 individual clones after the first and second round of selection, and allowed to bind to the wells of the microtiterplate.
  • MSA mouse serum albumin
  • a PCR was performed on positive clones after the second round of panning, with a set of primers binding to a sequence in the vector.
  • the PCR product was digested with the restriction enzyme Hinfl and loaded on a agarose gel. 4 clones were selected with a different Hinfl-pattern for further evaluation. Those clones were sequenced, and results are summarized in Table 4 (SEQ ID NOS: 1, 2 ,3 and 4).
  • the pellet was thawed at room temperature for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour.
  • Periplasmic fraction was isolated by centrifugation for 20 minutes at 4° C. at 20,000 rpm. The supernatant containing the nanobody was loaded on Ni-NTA and purified to homogeneity.
  • a microtiterplate was coated with 5 ⁇ g/ml MSA overnight at 4 C. After washing, the plate was blocked for 2 hours at RT with PBS-1% casein. Samples were applied in duplicate starting at a concentration of 2500 nM at 1 ⁇ 3 dilutions and allowed to bind for 2 hours at RT. A polyclonal rabbit anti-nanobody serum was added at 1/1000 (K208) for one hour at RT. Detection was with ant-rabbit alkaline phosphatase conjugate at 1/1000 and staining with PNPP as described in Example 6. Results are shown in FIG. 4 .
  • the E. coli production vector pAX11 was constructed to allow the two-step cloning of bivalent or bispecific VHH ( FIG. 5 ).
  • the carboxy terminal VHH was cloned first with Pstl and BstEll, while in the second step the other VHH was inserted by Sfil and Notl, which do not cut within the first gene fragment.
  • the procedure avoids the enforcement of new sites by amplification and thus the risk of introducing PCR errors.
  • the middle hinge of llama was used as a linker between the nanobodies.
  • a VHH against human TNF alpha was cloned at the COOH terminal of MSA specific nanobodies. Sequences are summarized in Table 4 (SEQ ID NOS: 5, 6, 7 and 8). Plasmid was prepared and was transformed into WK6 electrocompetent cells.
  • VHH against human TNF-alpha (MP7 12 b) is listed in Table 4 (SEQ ID NOS: 15, 16, 17 and 18).
  • a microtiter plate was coated with 5 ⁇ g/ml MSA overnight at 4° C. Plates were blocked for two hours at room temperature with 300 ⁇ l 1% casein in PBS. The plates were washed three times with PBS-Tween. Purified protein for the bispecific constructs was allowed to bind to the wells of the microtiterplate at a concentration of 0.4, 0.5, 2.5 and 2.5 ⁇ g/ml for MSA21, MSA24, MSA210 and MSA212 respectively. Plates were washed six times with PBS-Tween, Biotinilated TNF was added at a concentration of 10 ⁇ g/ml and diluted 3 fold, and allowed to bind for 2 hours at room temperature.
  • Binding was detected by incubation with mouse extravidin alkaline phosphatase conjugate (Sigma) 1/2000 in PBS, for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg 2 SO 4 , pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are shown in FIG. 6 and indicate that the bispecific construct can bind both antigens simultaneously.
  • PNPP p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg 2 SO 4 , pH9.8
  • Affinities for mouse albumin were determined in BIACORE by immobilization of mouse albumin on a CM5 BIAcore chip using EDC-NHS covalent coupling and are summarized in Table 5. The results indicate that the affinity for albumin is retained in the bispecific construct.
  • Pharamcokinetic experiments were initiated to compare half life in mice of the TNF-alpha binder TNF3E with MSA21/VHH#3E and MSA24/VHH#3E. Therefore our ELISA had to be optimized to obtain low background values when the samples are in blood or in plasma.
  • a microtiterplate was coated with neutravidin. After overnight incubation at 4 C, the plates were washed and blocked for 2 hours at RT with PBS-1% casein. 1 ⁇ g/ml biotinylated TNF-alpha was allowed to bind for 30 minutes at RT and the plate was washed.
  • mice CB57/BI6 for each construct were injected intravenously in the tail with 100 ⁇ g nanobody. Blood was retrieved at different time points (3 mice per time point) and serum was prepared. Samples were analyzed by ELISA for the presence of monovalent or bispecific nanobody as described in example 14. K208 was also compared to URL49 for the bispecific constructs to verify the integrity of the molecule. Results are shown in FIGS. 8 to 11 .
  • a trivalent nanobody was prepared by fusing the bivalent MSA21-MSA21 construct to target-specific nanobody TNF3E.
  • the resulting MSA21/MSA21/TNF3E (Table 7, and SEQ ID NO: 9) was tested in vivo according to the method of Example 16.
  • the library was prepared as described in Example 2.
  • the size of the library was 1.4 ⁇ 10 7 cfu, and >90% of the clones contained insert of the correct size. Phages were prepared as described in Example 3.
  • a well in a microtiterplate was coated with 2 ⁇ g/ml vWF or with PBS containing 1% casein. After overnight incubation at 4° C., the wells were blocked with PBS containing 1% casein, for 3 hours at RT. 200 ⁇ l phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10 ⁇ with PBS-Tween and 10 ⁇ with PBS. Phages were specifically eluted with 100 ⁇ l of 100 ⁇ g/ml collagen type III. Elutions were performed for overnight at room temperature.
  • a microtiter plate was coated overnight at 4° C. with collagen type III at 25 ⁇ g/ml in PBS. The plate was washed five times with PBS-Tween and blocked for 2 hours at room temperature with PBS containing 1% casein. The plate was washed five times with PBS-tween. 100 ⁇ l of 2 ⁇ g/ml vWF (vWF is pre-incubated at 37° C. for 15 minutes) was mixed with 20 ⁇ l periplasmic extract containing a VHH antibody (described in Example 6) and incubated for 90 minutes at room temperature in the wells of the microtiterplate. The plate was washed five times with PBS-tween.
  • DAKO anti-vWF-HRP monoclonal antibody
  • a microtiter plate was coated with 2 ⁇ g/ml vWF, overnight at 4° C. Plates were blocked for two hours at room temperature with 300 ⁇ l 1% casein in PBS. The plates were washed three times with PBS-Tween. Dilution series of all purified samples were incubated for 2 hours at RT. Plates were washed six times with PBS-Tween, after which binding of VHH was detected by incubation with mouse anti-myc mAB 1/2000 in PBS for 1 hour at RT followed by anti-mouse-HRP conjugate 1/1000 in PBS, also for 1 hour at RT. Staining was performed with the substrate ABTS/H 2 O 2 and the signals were measured after 30 minutes at 405 nm. The binding as a function of concentration of purified VHH is indicated in FIG. 12 .
  • Inhibition ELISA was performed as described in Example 20 but with decreasing concentrations of VHH and with human plasma at a dilution of 1/60 instead of with purified vWF. Results are represented in FIG. 13 .
  • the concentration of VHH resulting in 50% inhibition (IC50) is given in table 10.
  • Bispecific constructs were prepared with the first VHH specific for albumin (MSA21) and the second VHH specific for vWF. Constructs were made as described in Example 11. Sequences are shown in Table 4 (SEQ ID NOS: 19 to 21)
  • Protein was expressed and purified as described in Example 9. An extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%).
  • a microtiterplate was coated with 5 ⁇ g/ml mouse serum albumin overnight at 4° C. After washing the plate, wells were blocked for 2 hours with PBS-1% casein. The bispecific proteins were allowed to bind to the wells for 2 hours at RT. After washing, human, dog and pig plasma was added at different dilutions and allowed to bind for 2 hours at RT. Binding of vWF was detected with anti-vWF-HRP from DAKO at 1/3000 dilution. Staining was performed with ABTS/H 2 O 2 . Results are shown in FIG. 14 and indicate that functionality of both VHHs is retained in the bispecific construct.
  • F6 CDR3 Reverse primer Sfi1 (SEQ ID N° 45) GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCATGTGCAGCAAACC
  • F6 CDR3 Forward primer Not1 (SEQ ID N° 46) GTCCTCGCAACTGCGCGGCCGCCTGGCCCCAGAAGTCATACC
  • the PCR reactions was performed in 50 ml reaction volume using 50 pmol of each primer.
  • the reaction conditions for the primary PCR were 11 min at 94° C., followed by 30/60/120 sec at 94/55/72° C. for 30 cycles, and 5 min at 72° C. All reaction were performed wit 2.5 mM MgCl2 , 200 mM dNTP and 1.25 U AmpliTaq God DNA Polymerase (Roche Diagnostics, Brussels, Belgium).
  • the degree of amino acid sequence homology between anti-target single domain antibodies of the invention was calculated using the Bioedit Sequence Alignment Editor. The calculations indicate the proportion of identical residues between all of the sequences as they are aligned by ClustalW. (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research, submitted, June 1994).
  • Table 12 indicates the fraction homology between anti-serum albumin VHHs of the invention.
  • Table 13 indicates the fraction homology between anti-TNF-alpha VHHs of the invention.
  • Table 14 indicates the percentage homology between anti-IFN-gamma VHHs of the invention.
  • Table 15 indicates the fraction homology between anti-vWF VHHs of the invention.
  • Clones were selected after one and two rounds of selection and peripiasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 6.
  • IC50 values for bispecific nanobodies against albumin and against vWF as described in Example 28 IC50 (ng/ml) AM-2-75 100 MSA21/AM-2-75 60 AM-4-15-3 155 MSA21/AM-4-15-3 245 22-4L-16 100 MSA21/22-4L-16 140

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Diabetes (AREA)
  • Microbiology (AREA)
  • Pulmonology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Neurology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Vascular Medicine (AREA)
  • Endocrinology (AREA)

Abstract

The present invention relates to heterospecific polypeptide constructs comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target and at least one single domain antibody directed against a serum protein, said construct having a prolonged lifetime in biological circulatory systems. The invention further relates to methods for stabilising VHHs in biological circulatory systems.

Description

    FIELD OF THE INVENTION
  • The present invention provides heterospecific polypeptide constructs comprising one or more single domain antibodies, said constructs having improved stability in vivo and their use in diagnosis and therapy.
    • BACKGROUND OF THE INVENTION
  • Polypeptide therapeutics and in particular antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. However, in order to be effective as therapeutic agent, their pharmacokinetic profile should be optimized. The majority of current antibody applications are for acute disorders. There are however significant opportunities to develop antibody therapeutics for chronic conditions. This will require large doses of protein over a long period of time. Since the cost of antibody production in mammalian cells is high, the development of traditional antibody therapeutics for these applications has been discouraged. An alternative approach has been to express fragments of antibodies such as Fab's or single-chain Fv's in microbial expression systems such as yeast and bacteria. These fragments however have very short circulation times in vivo.
  • Some of the initial approaches to increase the circulation in the bloodstream of proteins and peptides were based on chemical modification, such as pegylation (U.S. Pat. No. 4,179,337). Examples of such products are PEG-Intron, i.e. pegylated interferon alpha-2b for the treatment of HCV, and treatment of chronic disorder with PEG-modified antibodies (A. P Chapman, Adv.Drug Delivery Reviews (2002), 54, 531-545). Such chemical methods, however, suffer from a number of disadvantages, such as inactivation of the target protein or peptide due to the chemical modification of certain amino acid side chains, instability of the target protein/peptide during the chemical reaction.
  • To overcome these limitations, alternative approaches have been developed, first of all by using non-conventional or modified proteins, secondly by using alternative methods to increase half-life in vivo. Stabilisation of the protein drug can therefore be carried out by choosing an inherently stable protein scaffold and providing methods to bind such scaffold to plasma proteins which occur in high concentrations, such as immunoglobulins or albumin. Binding to plasma protein can be an effective means to improving the pharmacokinetic properties of molecules in general. More precisely, binding to albumin to improve the half-life of proteins has been described: M. S. Dennis et al. (J. Biol. Chem. 33, 238390, 2002) isolated peptides having affinity for serum albumin. When bound to a Fab molecule, half-lives comparable to pegylated Fab's were obtained. Peptide ligands having affinity for IgG or serum albumin have been disclosed (WO 01/45746). Cemu Bioteknik (Nygren, Wigzell, Uhlen, EP 486525 B1; U.S. Pat. No. 6,267,964) described fusions of active proteins or peptides to polypeptides from bacterial origin that bind to serum albumin (e.g. Staph A). The drawback of these peptide-based approaches is that the peptides have to fold properly and be accessible to binding to serum albumin when fused to the therapeutic protein. Therefore, these peptides are inherently unstable and have affinities in the submicromolar range rather than subnanomolar or low nanomolar range, as is the case with conventional antibodies. As part of a larger protein, such as a conventional antibody molecule, binding of these peptides to albumin may be sterically hindered.
  • An alternative hybrid molecule with two functional units is based on a heterospecific antibody. Such a hybrid would consist of a bifunctional or heterospecific antibody construct with one entity having specificity and affinity for the target, the second entity having specificity and affinity for a serum protein, such as albumin. However, such heterospecific constructs based on conventional antibodies or Fab fragments have several important drawbacks: these are complex, large molecules composed of two polypeptide chains (VH and VL) and therefore difficult and expensive to produce in high amounts in mammalian expression systems. Furthermore, producing bifunctional antibodies composed of 4 chains (2 VH's and 2 VL's) have the inherent risk of resulting in molecules with the unproductive VH-VL combinations and consequent loss of activity. Several alternatives have been tried with mixed results based on peptide derivatives of conventional antibodies, such as diabodies and bifunctional scFv's (WO0220615; WO 9413804; WO 9119739; WO 9409131). Holliger et al (Nature Biotech. 15, 632-636, 1979) suggests that binding one of the antibody fragments of a diabody (bispecific construct derived from a conventional antibody) to serum immunoglobulin (IgG) may prolong serum residence time of such diabodies but no suggestion is made that bispecific diabodies may be stabilised using antibodies against a serum protein other than serum IgG. Diabodies are known to be inherently difficult to produce due to stickiness of their exposed surface and due to non-productive associations between the four different V-regions (2 VH+2 VL).
  • Covalent binding to serum proteins as disdosed in, for example, EP0793506B1, U.S. Pat. Nos. 5,612,034, 6,103,233, and US20020009441 using reactive groups forming stable covalent bonds to a serum protein or a cell have the inherent disadvantage of unwanted target modification through the reactive groups.
  • Fusions to large, long lived proteins such as albumin (Syed et al, Blood 89, 3243-3252 (1997), Yeh et al, PNAS 89, 1904-1908 (1992); Celltech (WO0027435)) or N-terminal fusions of albumin polypeptides (Delta Biotech/HGS, U.S. Pat. Nos. 5,380,712, 5,766,883) or the Fc portion of IgG (Capon et al, Nature 337, 525-531(1989); Ashkenazi et al, Curr. Op.Immunol. 9, 195-200 (1997)) have been described. Such fusions have the disadvantage of inefficient production and causing unwanted immunological reactions.
  • A complex of interferon with a monoclonal antibody to increase the serum half-life of interferon has been described in U.S. Pat. No. 5,055,289. Such approach has the inherent risk of impairing the biological activity of the interferon since the size of the construct raises the problem of steric hindrance.
    • THE AIMS OF THE PRESENT INVENTION
  • It is an aim of the present invention to provide therapeutic heterospecific antibody polypeptide constructs which overcome the problems of therapeutic antibodies of the art namely, low half-life in vivo, poor folding, low expression, and poor stability. It is a further aim of the present invention to provide methods for providing said heterospecific antibodies.
    • SUMMARY OF THE INVENTION
  • One-embodiment of the present invention is a polypeptide construct comprising:
      • at least one single domain antibody directed against a therapeutic and/or diagnostic target, and
      • at least one single domain antibody directed against a serum protein.
  • Another embodiment of the present invention is a polypeptide construct as described above wherein:
      • the number of anti-target single domain antibodies is at least two, and
      • at least two anti-target single domain antibodies do not share the same sequence, or all the anti-target single domain antibodies share the same sequence.
  • One embodiment of the present invention is a polypeptide construct as described above wherein:
      • the number of anti-serum protein single domain antibodies is at least two, and
      • at least two anti-serum-protein single domain antibodies do not share the same sequence, or all the anti-serum-protein single domain antibodies share the same sequence.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a Camelidae VHHs antibody.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a humanised Camelidae VHHs antibody.
  • One embodiment of the present invention is a polypeptide construct as described above wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a single domain anti-serum protein antibody correspond to a sequence represented by any of SEQ ID NOS: 1 to 4, and 28 to 40.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is TNF-alpha-alpha.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.
  • One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said disorders are any of rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is vWF
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is collagen.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least one anti-target single domain antibody is anti-vWF VHHs.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NOS: 19 to 21.
  • One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is IgE.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least anti-target single domain antibody is anti-IgE VHHs.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NOS: 22 to 24.
  • One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a polypeptide construct as described above wherein a target is IFN-gamma.
  • One embodiment of the present invention is a polypeptide construct as described above wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.
  • One embodiment of the present invention is a polypeptide construct as described above corresponding to a sequence represented by SEQ ID NOS: 25 to 27.
  • One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a polypeptide construct as described above directed against a single target wherein said target is involved in a disease process.
  • One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion thereof, of an homologous sequence of a functional portion thereof.
  • One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic add as described above for use in the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.
  • One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time.
  • One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is remains active in the circulation for extended periods of time.
  • One embodiment of the present invention is a polypeptide construct or nucleic acid as described above, or use of a polypeptide construct or nucleic acid as described above, wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
  • One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid as described above and a pharmaceutically acceptable vehicle.
  • One embodiment of the present invention is a method of producing a as described above comprising
  • (a) culturing host cells comprising nucleic acid capable of encoding a polypeptide as described above, under conditions allowing the expression of the polypeptide, and,
  • (b) recovering the produced polypeptide from the culture.
  • One embodiment of the present invention is a method as described above, wherein said host cells are bacterial or yeast.
  • One embodiment of the present invention is a method for prolonging the half-life of a single domain antibody in the blood stream of a subject, said antibody directed against a therapeutic and/or diagnostic target by joining thereto one or more single domain antibodies directed against a serum protein.
  • One embodiment of the present invention is a method as described above wherein said anti-target single domain antibodies do not share the same sequence.
  • One embodiment of the present invention is a method as described above wherein said anti-serum protein single domain antibodies do not share the same sequence.
  • One embodiment of the present invention is a method as described above wherein said single domain antibodies are Camelidae VHH antibodies.
  • One embodiment of the present invention is a method as described above wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.
  • One embodiment of the present invention is a method as described above wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOS: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • One embodiment of the present invention is a composition comprising a polypeptide as described above or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.
    • BRIEF DESCRIPTION OF FIGURES AND TABLES
  • FIG. 1 phage ELISA to show that HSA-specific nanobodies are present in the library as described in Example 4.
  • FIG. 2 Binding of phages expressing the albumin binders, to plasma blotted on nitrocellulose as described in Example 8.
  • FIG. 3 Coomassie staining of plasma samples on SDS-PAGE as described in example 8.
  • FIG. 4 Binding of purified nanobodies to mouse albumin as determined by ELISA as described in Example 10.
  • FIG. 5 Multiple cloning site of PAX011 for construction of bispecific nanobodies as described in Example 11.
  • FIG. 6 Sandwich ELISA to show the functionality of both nanobodies in the bispecific construct as described in Example 12.
  • FIG. 7 Optimization of ELISA to determine nanobody concentration in 10% plasma or in 10% blood as described in Example 14.
  • FIG. 8 Pharmacokinetics for the monovalent anti-TNF-α nanobody in mice as determined by ELISA as described in Example 16.
  • FIG. 9 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA as described in Example 16.
  • FIG. 10 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA with K208 as compared to URL49 as described in Example 16.
  • FIG. 11 Pharmacokinetics for the bispecific nanobody MSA24/TNF3E in mice as determined by ELISA as described in Example 16.
  • FIG. 12 Binding to vWF as determined by ELISA, by purified VHH as described in Example 23.
  • FIG. 13 ELISA to test inhibition by VHH of binding of vWF to collagen as described in Example 24.
  • FIG. 14 Sandwich ELISA showing the functionality of both VHHs in a bispecific construct as described in example 27.
  • Table 1 Immunization scheme according to Example 1
  • Table 2 Results after one and two rounds of panning on mouse serum albumin as described in example 5.
  • Table 3 Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 6.
  • Table 4 Sequence listing
  • Table 5 Affinities (koff, kon and KD) for albumin binders as determined by BIACORE as described in Example 13.
  • Table 6 Results for the LAL-assay for monovalent and bispecific nanobodies after purification on polymyxin as described in Example 15.
  • Table 7 Immunization scheme used for llama 002 according to Example 17.
  • Table 8 Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in example 19. Pfu vWF (antigen) divided by pfu casein (a specific binding)=enrichment.
  • Table 9 Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 20.
  • Table 10 Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 23.
  • Table 11 IC50 values for bispecific nanobodies against albumin and against vWF as described in Example 28.
  • Table 12 Fractional homologies between the amino acid sequences of anti-mouse serum albumin VHHs of the invention.
  • Table 13 Fractional homologies between anti-TNF-alpha VHHs of the invention.
  • Table 14 Percentage homologies between anti-IFN-gamma VHHs of the invention.
  • Table 15 Fractional homologies between anti-vWF VHHs of the invention.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention relates to a heterospecific polypeptide construct comprising one or more single domain antibodies each directed against a serum protein(s) of a subject, and one or more single domain antibodies each directed against a target molecule(s) and the finding that the construct has a significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target single domain antibody when not part of such a construct.
  • Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678 for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.
  • The one or more single domain antibodies of the polypeptide construct which are directed against a target may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-target single domain antibodies which do not all share the same sequence, but which are directed against the same target, or fragment thereof, one or more antigens thereof.
  • In accordance with the present invention there are provided methods for the utilization of a plurality of anti-target and/or anti-serum protein single domain antibodies to increase the avidity and/or affinity of the heterospecific molecule. In this manner, serum half-lives of molecules modified in accordance with the invention can be extended. Such modification will modify and/or extend the therapeutic window of a specific therapeutic molecule. This flexibility cannot be achieved with alternative methods in the art, such as when using peptides with specificity to serum proteins, diabodies which are difficult to produce in a multivalent form, chemical modifications (such as pegylation, acylation).
  • The one or more single domain antibodies of the polypeptide construct which are directed against a serum protein may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-serum protein single domain antibodies which do not all share the same sequence, but which are directed against serum protein, or fragment thereof, one or more antigens thereof.
  • In another embodiment, one or more anti-target single domain antibodies of the polypeptide construct may be directed to more than one target (e.g. vWF and collagen). Similarly, the anti-serum protein single domain antibodies of the polypeptide construct may be directed against more than one serum protein (e.g. serum albumin and fibrinogen).
  • VHHs, according to the present invention, and as known to the skilled addressee are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelids as described in WO9404678 (and referred to hereinafter as VHH domains or nanobodies). VHH molecules are about 10× smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Camelids (WO 9749805). As such, anti-albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein. As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into ‘unusual’ or non-conventional epitopes such as cavities (WO9749805), the affinity of such VHH's to circulating albumin may be increased.
  • The present invention also relates to the finding that a heterospecific polypeptide construct comprising one or more VHHs directed against one or more serum proteins of a subject, and one or more VHHs directed against one or more target molecule of said subject surprisingly has significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target VHH when not part of said construct. Furthermore, such prolonged half-life is in the range of several days due to the high affinity anti-serum albumin VHH's compared to several hours when using low affinity peptides specific for albumin (Dennis et al, JBC, 277, 35035). The extension of the half-life is demonstrated by the inventors herein, for example, in Example 16, and by the polypeptide represented by SEQ ID NO: 5. Furthermore, the said construct was found to exhibit the same favourable properties of VHHs such as high stability remaining intact in mice for at least 19 days (Example 16), extreme pH resistance, high temperature stability and high target affinity.
  • A target according to the invention is any biological substance capable of binding to a heterospecific polypeptide construct of the invention. Targets may be, for example, proteins, peptides, nucleic acids, oligonucleic acids, saccharides, polysaccharides, glycoproteins. Examples include, but are not limited to therapeutic targets, diagnostic targets, receptors, receptor ligands, viral coat proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins, or a fragment thereof. Targets may be native protein or a fragment thereof, a homologous sequence thereof, a functional portion thereof, or a functional portion of an homologous sequence.
  • The properties of single domain antibodies, in particular VHHs, compare favourably with those of antibodies derived from sources such as mouse, sheep, goat, rabbit etc. (i.e. traditional antibodies), and humanised derivatives thereof. Traditional antibodies are not stable at room temperature, and have to be refrigerated for preparation and storage, requiring necessary refrigerated laboratory equipment, storage and transport, which contribute towards time and expense. Refrigeration is sometimes not feasible in developing countries. Furthermore, the manufacture or small-scale production of said antibodies is expensive because the mammalian cellular systems necessary for the expression of intact and active antibodies require high levels of support in terms of time and equipment, and yields are very low. Furthermore, traditional antibodies have a binding activity which depends upon pH, and hence are unsuitable for use in environments outside the usual physiological pH range such as, for example, in treating gastric bleeding, gastric surgery. Furthermore, traditional antibodies are unstable at low or high pH and hence are not suitable for oral administration.
  • However, it has been demonstrated that VHHs resist harsh conditions, such as extreme pH, denaturing reagents and high temperatures (Ewert S et al, Biochemistry 2002 Mar 19;41 (11):3628-36), so making them suitable for delivery by oral administration. Furthermore, traditional antibodies have a binding activity which depends upon temperature, and hence are unsuitable for use in assays or kits performed at temperatures outside biologically active-temperature ranges (e.g. 37±20° C.).
  • Furthermore VHHs are more soluble, meaning they may be stored and/or administered in higher concentrations compared with conventional antibodies. The polypeptides of the present invention also retain binding activity at a pH and temperature outside those of usual physiological ranges, which means they may be useful in situations of extreme pH and temperature which require a modulation of platelet-mediated aggregation, such as in gastric surgery, control of gastric bleeding, assays performed at room temperature etc. The polypeptides of the present invention also exhibit a prolonged stability at extremes of pH, meaning they would be suitable for delivery by oral administration. The polypeptides of the present invention may be cost-effectively produced through fermentation in convenient recombinant host organisms such as Escherichia coli and yeast; unlike conventional antibodies which also require expensive mammalian cell culture facilities, achievable levels of expression are high. Examples of yields of the polypeptides of the present invention are 1 to 10 mg/ml (E. coli) and up to 1 g/l (yeast). The polypeptides of the present invention also exhibit high binding affinity for a broad range of different antigen types, and ability to bind to epitopes not recognised by conventional antibodies; for example they display long CDR-based loop structures with the potential to penetrate into cavities and exhibit enzyme function inhibition. Furthermore, since binding often occurs through the CDR3 loop only, it is envisaged that peptides derived from CDR3 could be used therapeutically (Desmyter et al, J Biol Chem, 2001, 276: 26285-90). The polypeptides of the invention are also able to retain full binding capacity as fusion protein with an enzyme or toxin.
  • The present invention also relates to a heterospecific polypeptide construct comprising one or more VHHs each directed against one or more serum proteins of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to the traditional class of Camelidae single domain heavy chain antibodies. The present invention also relates to a heterospecific polypeptide construct comprising one or more VHH each directed against one or more serums protein of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to a class of Camelidae single domain heavy chain antibodies that have human-like sequences. A VHH sequence represented by SEQ ID NO: 12 which binds to TNF-alpha and a second VHH which binds to mouse albumin, belongs to this class of VHH peptides. As such, peptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to patients directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.
  • A human-like class of Camelidae single domain antibodies represented by SEQ ID No. 1, 3 and 4 have been described in WO03035694 and contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.
  • Therefore, one aspect of the present invention allows for the direct administration of an anti-serum albumin polypeptide, wherein the single domain antibodies belong to the humanized class of VHH, and comprise a sequence represented by any of SEQ ID NO: 1, 3 or 4 to a patient in need of the same.
  • A subject as used herein is any mammal having a circulatory system in which the fluid therein comprises serum proteins. Examples of circulatory system include blood and lymphatic systems. Examples of animals include, but are not limited to, rabbits, humans, goats, mice, rats, cows, calves, camels, llamas, monkeys, donkeys, guinea pigs, chickens, sheep, dogs, cats, horses etc.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target, and at least one single domain antibodies each directed against one or more serum proteins or polypeptides. As already mentioned, the anti-target single domain antibodies may have the same sequence. Alternatively, at least two anti-target single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same target, fragments thereof, or antigen thereof. Similarly, the anti-serum protein single domain antibodies may have the same sequence. Alternatively, at least two anti-serum protein single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same serum protein, fragments thereof, or antigen thereof.
  • In another embodiment of the present invention, where more than one anti-target single domain antibodies is present in the heterospecific polypeptide construct, each anti-target single domain antibody may be directed to a different target (e.g. one to vWF and one to collagen). Similarly, where more than one anti-serum protein single domain antibody is present, each anti-serum single domain antibody may be directed to a different serum protein (e.g. one to serum albumin and one to fibrinogen).
  • One embodiment of the invention, is a heterospecific polypeptide, wherein an anti-serum protein single domain antibody corresponds to a sequence represented by any of SEQ ID NOS:1 to 4 and 28 to 40.
  • The constructs disclosed herein retain the advantageous properties of single domain antibodies (e.g. VHHs) and have a prolonged lifetime in the circulation of an individual. Thus, such constructs are able to circulate in the subject's serum for several days, reducing the frequency of treatment, the inconvenience to the subject and resulting in a decreased cost of treatment. Furthermore, it is an aspect of the invention that the half-life of the heterospecific polypeptide constructs may be controlled by the number of anti-serum protein single domain antibodies present in the construct. A controllable half-life is desirable in several circumstances, for example, in the application of a timed dose of a therapeutic heterospecific polypeptide construct, or to obtain a desired therapeutic effect.
  • According to an aspect of the Invention a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a homologous sequence of a full-length heterospecific polypeptide construct. According to an aspect of the invention a heterospecific polypeptide construct may comprise a sequence of a heterospecific polypeptide construct.
  • According to an aspect of the invention a single domain antibody used to form a heterospecific polypeptide construct may be a complete single domain antibody (e.g. a VHH) or a homologous sequence thereof. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a homologous sequence of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a homologous sequence of a complete single domain antibody.
  • According to another aspect of the invention a heterospecific polypeptide construct may be an homologous sequence of the parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a homologous sequence of the parent sequence.
  • As used herein, an homologous sequence of the present invention may comprise additions, deletions or substitutions of one or more amino acids, which do not substantially alter the functional characteristics of the polypeptides of the invention. The number of amino acid deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • A homologous sequence of the present invention may include a single domain antibody of the invention which has been humanised.
  • By humanised is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent. Humanising a single domain antibody, according to the present invention, comprises a step of replacing one or more of amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanisation does not significantly affect the antigen binding capacity of the resulting polypeptide. Such methods are known by the skilled addressee. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of any of the following residues either alone or in combination: some VHH contain typical Camelidae hallmark residues at position 37, 44, 45 and 47 with hydrophilic characteristics. Replacement of the hydrophilic residues by human hydrophobic residues at positions 44 and 45 (E44G and R45L) did not have an effect on binding and/or inhibition. Further humanization may be required by substitution of residues in FR 1, such as position 1, 5, 28 and 30; FR3, such as positions 74, 75, 76, 83, 84, 93 and 94; and FR4, such as position 103, 104, 108 and 111 (all numbering according to the Kabat).
  • One embodiment of the present invention is a method for humanizing a VHH comprising the steps of replacing of any of the following residues either alone or in combination:
      • FR1 position 1, 5, 28 and 30,
      • the hallmark amino acid at position 44 and 45 in FR2,
      • FR3 residues 74, 75, 76, 83, 84, 93 and 94,
      • and positions 103, 104, 108 and 111 in FR4;
        (numbering according to the Kabat numbering).
  • Some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanisation. Therefore, one aspect of the present invention allows for the formation of a heterospecific polypeptide construct without humanisation of the VHH, when said VHH exhibit high homology to human VH framework regions.
  • A homologous sequence of the present invention may be a sequence of the invention derived from another species such as, for example, camel, llama, dromedary, alpaca, guanaco etc.
  • Where homologous sequence indicates sequence identity, it means a sequence which presents a high sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with a single domain antibody of the invention, and is preferably characterised by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.
  • A homologous sequence according to the present invention may refer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000 nucleotides able to hybridise to the reverse-complement of the nucleotide sequence capable of encoding a native sequence under stringent hybridisation conditions (such as the ones described by SAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York).
  • As used herein, a functional portion refers to a single domain antibody of sufficient length such that the interaction of interest is maintained with affinity of 1×10−6 M or better.
  • Alternatively a functional portion of a single domain antibody of the invention comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target or serum protein.
  • As used herein, a functional portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.
  • A portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.
  • Targets as mentioned herein such as TNF-alpha, IFN-gamma receptor, serum proteins (e.g. serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, fibrinogen) and IFN-gamma may be fragments of said targets. Thus a target is also a fragment of said target, capable of eliciting an immune response. A target is also a fragment of said target, capable of binding to a single domain antibody raised against the full length target.
  • A fragment as used herein refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is of sufficient length such that the interaction of interest is maintained with affinity of 1×10−6 M or better.
  • A fragment as used herein also refers to optional insertions, deletions and substitutions of one or more amino acids which do not substantially alter the ability of the target to bind to a single domain antibody raised against the wild-type target. The number of amino acid insertions deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • The serum protein may be any suitable protein found in the serum of subject, or fragment thereof. In one aspect of the invention, the serum protein is serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen. Depending on the intended use such as the required half-life for effective treatment and/or compartimentalisation of the target antigen, the VHH-partner can be directed to one of the above serum proteins.
  • A single domain antibody directed against a target means single domain antibody that it is capable of binding to its target with an affinity of better than 10−6 M.
  • The heterospecific polypeptide constructs disclosed herein may be made by the skilled artisan according to methods known in the art or any future method. For example, VHHs may be obtained using methods known in the art such as by immunising a camel and obtaining hybridomas therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by using phage display.
  • The anti-serum protein single domain antibody may be directed against a polypeptide of a serum protein or a whole protein. The anti-target single domain antibody may be directed against a polypeptide of said target of the whole target. Methods for scanning a protein for immunogenic polypeptides are well known in the art.
  • The single domain antibodies may be joined using methods known in the art or any future method. For example, they may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the single domain antibody may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more anti-target single domain antibodies and one or more anti-serum protein single domain antibodies. A method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO 96/34103. One way of joining multiple single domain antibodies is via the genetic route by linking single domain antibody coding sequences either directly or via a peptide linker. For example, the C-terminal end of the first single domain antibody may be linked to the N-terminal end of the next single domain antibody. This linking mode can be extended in order to link additional single domain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.
  • An aspect of the present invention is the administration of heterospecific polypeptide constructs according to the invention which avoids the need for injection. Conventional antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity, however, they have one important drawback: these are complex, large molecules and therefore relatively unstable, and they are sensitive to breakdown by proteases. This means that conventional antibody drugs cannot be administered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation because they are not resistant to the low pH at these sites, the action of proteases at these sites and in the blood and/or because of their large size. They have to be administered by injection (intravenously, subcutaneously, etc.) to overcome some of these problems. Administration by injection requires specialist training in order to use a hypodermic syringe or needle correctly and safely. It further requires sterile equipment, a liquid formulation of the therapeutic polypeptide, vial packing of said polypeptide in a sterile and stable form and, of the subject, a suitable site for entry of the needle. Furthermore, subjects commonly experience physical and psychological stress prior to and upon receiving an injection. An aspect of the present invention overcomes these problems of the prior art, by providing the heterospecific polypeptides constructs of the present invention. Said constructs are sufficiently small, resistant and stable to be delivered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation substantial without loss of activity. The heterospecific polypeptides constructs of the present invention avoid the need for injections, are not only cost/time savings, but are also more convenient and more comfortable for the subject.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.
  • As known by persons skilled in the art, once in possession of said polypeptide construct, formulation technology may be applied to release a maximum amount of VHHs in the right location (in the stomach, in the colon, etc.). This method of delivery is important for treating, prevent and/or alleviate the symptoms of disorder whose targets that are located in the gut system.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder susceptible to modulation by a therapeutic compound that is able pass through the gastric environment without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the gut system without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract.
  • In a non-limiting example, a formulation according to the invention comprises a heterospecific polypeptide construct as disclosed herein comprising one or more VHHs directed against one or more targets in the form of a gel, cream, suppository, film, or in the form of a sponge or as a vaginal ring that slowly releases the active ingredient over time (such formulations are described in EP 707473, EP 684814, U.S. Pat. No. 5,629,001).
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound to the vaginal and/or rectal tract, by vaginally and/or rectally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the vaginal and/or rectal tract without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target comprising at least one single domain antibody directed against a target, for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung.
  • In a non-limiting example, a formulation according to the invention, comprises a heterospecific polypeptide construct as disclosed herein directed against one or more targets in the form of a nasal spray (e.g. an aerosol) or inhaler. Since the construct is small, it can reach its target much more effectively than therapeutic IgG molecules.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound delivered to the upper respiratory tract and lung, by administering to a subject a heterospecific polypeptide construct as disclosed herein wherein one or more single domain antibodies are specific for an antigen related to the disorder, by inhalation through the mouth or nose.
  • Another aspect of the invention is a dispersible VHH composition, in particular dry powder dispersible VHH compositions, such as those described in U.S. Pat. No. 6,514,496. These dry powder compositions comprise a plurality of discrete dry particles with an average particle size in the range of 0.4-10 mm. Such powders are capable of being readily dispersed in an inhalation device. VHH's are particularly suited for such composition as lyophilized material can be readily dissolved (in the lung subsequent to being inhaled) due to its high solubilisation capacity (Muyldermans, S., Reviews in Molecular Biotechnology, 74, 277-303, (2001)). Alternatively, such lyophilized VHH formulations can be reconstituted with a diluent to generate a stable reconstituted formulation suitable for subcutaneous administration. For example, anti-IgE antibody formulations (Example 1; U.S. Pat. No. 6,267,958, EP 841946) have been prepared which are useful for treating allergic asthma.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung without being inactivated.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and lung, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and/or lung without being inactivated, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • One embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa. Because of their small size, a heterospecific polypeptide construct as disclosed herein can pass through the intestinal mucosa and reach the bloodstream more efficiently in subjects suffering from disorders which cause an increase in the permeability of the intestinal mucosa.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa, by orally administering to a subject a heterospecific polypeptide construct as disclosed herein.
  • This process can be even further enhanced by an additional aspect of the present invention—the use of active transport carriers. In this aspect of the invention, VHH is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this “carrier” is a second VHH which is fused to the therapeutic VHH. Such fusion constructs are made using methods known in the art. The “carrier” VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the intestinal mucosa without being inactivated, by administering orally to a subject a heterospecific polypeptide construct of the invention.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering orally to a subject a heterospecific polypeptide construct of the invention.
  • This process can be even further enhanced by an additional aspect of the present invention—the use of active transport carriers. In this aspect of the invention, a heterospecific polypeptide construct as described herein is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this “carrier” is a VHH which is fused to said polypeptide. Such fusion constructs made using methods known in the art. The “carriers” VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the tissues beneath the tongue effectively. A formulation of said polypeptide construct as disclosed herein, for example, a tablet, spray, drop is placed under the tongue and adsorbed through the mucus membranes into the capillary network under the tongue.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound that is able pass through the tissues beneath the tongue effectively, by sublingually administering to a subject a VHH specific for an antigen related to the disorder.
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able to pass through the tissues beneath the tongue.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the tissues beneath the tongue without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the skin effectively. A formulation of said polypeptide construct, for example, a cream, film, spray, drop, patch, is placed on the skin and passes through.
  • An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound that is able pass through the skin effectively, by topically administering to a subject a heterospecific polypeptide construct as disclosed herein comprising one or more single domain antibodies specific for an antigen related to the disorder.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct as disclosed herein as a topical ophthalmic composition for the treatment of ocular disorder, such as allergic disorders, which method comprises the topical administration of an ophthalmic composition comprising polypeptide construct as disclosed herein, said construct comprising one or more anti-IgE VHH (Example 1, Example 2).
  • Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the skin effectively.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the skin without being inactivated, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.
  • In another embodiment of the present invention, a heterospecific polypeptide construct further comprises a carrier single domain antibody (e.g. VHH) which acts as an active transport carrier for transport said heterospecific polypeptide construct, the lung lumen to the blood.
  • A polypeptide construct further comprising a carrier binds specifically to a receptor present on the mucosal surface (bronchial epithelial cells) resulting in the active transport of the polypeptide from the lung lumen to the blood. The carrier single domain antibody may be fused to the polypeptide construct. Such fusion constructs made using methods known in the art and are describe herein. The “carrier” single domain antibody binds specifically to a receptor on the mucosal surface which induces an active transfer through the surface.
  • Another aspect of the present invention is a method to determine which single domain antibodies (e.g. VHHs) are actively transported into the bloodstream upon nasal administration. Similarly, a naïve or immune VHH phage library can be administered nasally, and after different time points after administration, blood or organs can be isolated to rescue phages that have been actively transported to the bloodstream. A non-limiting example of a receptor for active transport from the lung lumen to the bloodstream is the Fc receptor N (FcRn). One aspect of the invention includes the VHH molecules identified by the method. Such VHH can then be used as a carrier VHH for the delivery of a therapeutic VHH to the corresponding target in the bloodstream upon nasal administration.
  • One embodiment of the present invention is a heterospecific polypeptide construct for use in treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound intraveneously. An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound via the bloodstream.
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation. An aspect of the invention is the use of a said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof. By using a heterospecific polypeptide construct of the present invention to treat or diagnose an aforementioned disorder, the depletion of said construct is retarded.
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound that is able to circulate in the patients serum for several days, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof. By using a heterospecific polypeptide construct of the present invention to treat or diagnose an aforementioned disorder, the frequency of treatment is reduced, so resulting in a decreased cost of treatment.
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder relating to allergies. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • The above aspects and embodiments of the invention also apply when an anti-serum single domain antibody of the aforementioned heterospecific polypeptide constructs corresponds to a sequence represented by SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.
  • The above aspects and embodiments of the invention also apply when a heterospecific polypeptide construct of the invention corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-TNF-alpha Camelidae VHH.
  • The above aspects and embodiments of the invention also apply when an heterospecific polypeptide constructs of the invention corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21 a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-vWF Camelidae VHH.
  • The above aspects and embodiments of the invention also apply when an heterospecific polypeptide constructs of the invention corresponds to a sequence represented by any of SEQ ID NOS: 22 to 24 a homologous sequence thereof, a functional portion thereof. Said sequences comprise an anti-IgE Camelidae VHH.
  • The above aspects and embodiments of the invention also apply when an heterospecific polypeptide construct according to the invention corresponds to a sequence represented by any of SEQ ID NOS:25 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-Interferon-gamma Camelidae VHH.
  • A non-limiting example, in relation to allergies, of a target against which an anti-target single domain antibody may be directed is IgE. During their lifetime, subjects can develop an allergic response to harmless parasites such as Dermatophagoides pteronyssinus, the house dust mite or to substances such as clumps, plastics, metals. This results in an induction of IgE molecules that initiates a cascade of immunological responses. One aspect of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies. In one aspect of the invention, said anti-IgE single domain antibodies prevents the interaction of IgE with their receptor(s) on mast cells and basophils, so blocking initiation of the immunological cascade and a subsequent allergic reaction. In another aspect an anti-serum protein single domain antibody is directed to one of the subject's serum proteins. A heterospecific polypeptide construct as disclosed herein thus reduces or prevents an allergic response due to common or unusual allergens. Furthermore, the construct has a prolonged lifetime in the blood so increasing the therapeutic window. 7
  • Tumor necrosis factor alpha (TNF-alpha) is believed to play an important role in various diseases, for example in inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNF-alpha and the receptors (CD120a, CD120b) have been studied in great detail. TNF-alpha in its bioactive form is a trimer and the groove formed by neighboring subunits is important for the cytokine-receptor interaction. Several strategies to antagonize the action of the cytokine have been developed and are currently used to treat various disease states.
  • A TNF inhibitor which has sufficient specificity and selectivity to TNF may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory diseases. However, it is extremely difficult and a lengthy process to develop a small chemical entitly (NCE) with sufficient potency and selectivity to such target sequence. Antibody-based therapeutics on the other hand have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. In addition, the development time can be reduced considerably when compared to the development of new chemical entities (NCE's). However, conventional antibodies are difficult to elicit against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with TNF-alpha.
  • The heterospecific polypeptide constructs of the present invention, wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties such as stability, size, and reliable expressioin. Furthermore, the inventors have found that, despite presence of a groove in multimeric TNF-alpha, the heterospecific polypeptide constructs are still able to achieve strong binding to TNF-alpha
  • Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, an anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.
  • According to one aspect of the invention, a target against which a single domain antibody of a heterospecific polypeptide construct is directed is tumor necrosis factor alpha (TNF-alpha). TNF-alpha is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • Anti-target single domain antibodies may be directed against whole TNF-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibody fused to one or more anti-serum protein single domain antibody, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 5 to 18. The anti-TNF-alpha single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to TNF-alpha.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of inflammatory disorders. TNF-alpha is involved in inflammatory processes, and the blocking of TNF-alpha action can have an anti-inflammatory effect, which is highly desirable in certain disorder states such as, for example, Crohn's disease. Oral delivery of these heterospecific polypeptide construct results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain TNF-alpha producing cells. These heterospecific polypeptide constructs can neutralise the TNF-alpha locally, avoiding distribution throughout the whole body and thus limiting negative side-effects. Genetically modified microorganisms such as Micrococcus lactis are able to secrete antibody fragments. Such modified microorganisms can be used as vehicles for local production and delivery of antibody fragments in the intestine. By using a strain which produces a TNF-alpha-neutralising heterospecific polypeptide construct, inflammatory bowel disorder could be treated.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.
  • It is an aspect of the invention that the anti-TNF-alpha single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 5 to 18 comprise anti-TNF alpha Camelidae VHH and anti-mouse serum albumin Camelidae VHH.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (anti-serum protein Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • The inventors have found that a heterospecific polypeptide construct comprising a sequence corresponding to any of SEQ ID NOS: 5 to 18 surprisingly exhibits higher than expected affinity towards its target and prolonged half-life in the circulatory system.
  • Platelet-mediated aggregation is the process wherein von Willebrand Factor (vWF)-bound collagen adheres to platelets and/or platelet receptors (examples of both are gpla/lla, gplb, or collagen), ultimately resulting in platelet activation. Platelet activation leads to fibrinogen binding, and finally to platelet aggregation. The ability to disrupt platelet-mediated aggregation has many applications including the treatment of disease as mentioned below. Since the heterospecific polypeptide constructs of the invention effective prevent clotting, and the half-life thereof is controllable, they may be used for surgical procedures, for example, which require an inhibition of platelet-mediated aggregation for a limited time period.
  • Monovalent single domain antibodies such as VHHs show surprisingly high platelet aggregation inhibition in experiments to measure platelet aggregation inhibition under high shear: 50% inhibition of platelet aggregation was obtained at a concentration between 4 and 25 nM. In comparison, the Fab fragment derived from a vWF-specific antibody inhibiting the interaction with collagen, 82D6A3, inhibits 50% of platelet aggregation at approximately a twenty-fold higher concentration (Vanhoorelbeke K. et al, Journal of Biological Chemistry, 2003, 278: 37815-37821). These results were unexpected given that the IC50 values for the monovalent VHH's are up to 225 times fold worse in ELISA then the IC50 value of the IgG of 82D6A3.
  • This clearly shows that IgG antibodies is not suited to interaction with macromolecules which are starting, or are in the process of aggregating, such as those involved in platelet-mediated aggregation. vWF makes multimers of up to 60 monomers (final multimers of up to 20 million dalton in size). Indeed, it has been shown that not all A3 domains are accessible to 82D6A3 (Dongmei WU, Blood, 2002, 99, 3623 to 3628). Furthermore the large size of conventional antibodies, would restrict tissue penetration, for example, during platelet-mediated aggregation at the site of a damaged vessel wall.
  • The structure of single domain antibodies, in particular is unique. For example VHH molecules derived from Camelidae antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense tissues and for accessing the limited space between macromolecules participating in or starting the process of platelet mediated aggregation.
  • To our knowledge, this is the first time that experiments show, that the small size of a VHH is advantageous over a large intact antibody for inhibition of interactions between such large macromolecules.
  • Despite the small size of nanobodies, and thus advantages for penetration, it is still surprising that such a small molecule can inhibit interactions between large polymers such as vWF (up to 60 monomers) and collagen and with such a high efficiency. It has been described that only the large multimeric forms of vWF are hemostatically active (Furian, M,. 1996, Ann. Hematol. 72:341-348). Binding of multimeric vWF to collagen occurs with ˜100-fold higher affinity than binding of monomeric vWF fragments.
  • The results from the high shear experiments indicate that a lower dose will be needed for administration to patients. Therefore, fewer side effects are expected (such as immunogenicity or bleeding problems).
  • It is an aspect of the present invention to provide heterospecific polypeptide constructs which modulate processes which comprise platelet-mediated aggregation such as, for example, vWF-collagen binding, vWF-platelet receptor adhesion, collagen-platelet receptor adhesion, platelet activation, fibrinogen binding and/or platelet aggregation. Said heterospecific polypeptide constructs are derived from single domain antibodies directed towards vWF, vWF A1 or A3 domains, gplb or collagen.
  • Anti-target single domain antibodies may be directed against whole vWF, vWF A1 or A3 domains, gplb or collagen or a fragment thereof, or a fragment of a homologous sequence thereof.
  • According to one aspect of the invention, a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is von Willebrand factor (vWF). According to another aspect of the invention, the target is vWF A1 or A3 domains. According to another aspect of the invention, the target is gplb. According to another aspect of the invention, the target is gpla/llA. According to another aspect of the invention, the target is collagen.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies fused to one or more anti-serum protein VHHs, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 19 to 21. The anti-vWF single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to vWF.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen for use in treating, preventing and/or alleviating the symptoms of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof. Said disorders include transient cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis. Said conditions include those arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.
  • One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders or conditions relating to relating to platelet-mediated aggregation or dysfunction thereof, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.
  • It is an aspect of the invention that the anti-vWF, anti-vWF A1 or anti-vWF A3 or anti-collagen VHHs of the present invention may be derived from VHHs of any class. For example, they may be derived from the class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 19 to 21 comprise anti-vWF VHH and anti-mouse serum albumin VHH.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen and wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • During their lifetime, subjects may develop an allergic response to harmless parasites (e.g. Dermatophagoides pteronyssinus, house dust mite) or substances (clumps, plastics, metals). This results in the induction of IgE molecules that initiate a cascade of immunological responses. One aspect of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies, said heterospecific polypeptide construct preventing the interaction of IgEs with their receptor(s) on mast cells and basophils. As such they prevent the initiation of the immunological cascade, an allergic reaction.
  • According to one aspect of the invention, a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is IgE. Said antibodies may be directed against whole IgE or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 22 to 24. The anti-IgE single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IgE.
  • Anti-target single domain antibodies may be directed against whole IgE-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibody fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of disorders relating to allergies. Said disorders comprise a wide range of IgE-mediated diseases such as hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.
  • One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said VHH is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to allergies, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.
  • Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.
  • It is an aspect of the invention that the anti-IgE single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. Said VHHs may be derived from Camelidae. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the heterospecific polypeptides correspond to a sequence represented by any of SEQ ID NOS: 22 to 24 , a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 22 to 24 comprise anti-IgE Camelidae VHH and anti-mouse serum albumin Camelidae VHH.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said ant-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (ant-protein serum Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • A heterospecific polypeptide construct as disclosed herein prevents thus reduces or prevents an allergic response due to common or unusual allergens. Furthermore, the construct has a prolonged lifetime in the blood so increasing the therapeutic window.
  • Interferon gamma (IFN-gamma) is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, multiple sclerosis and hyperimmune reactions in the eye. IFN-gamma has also been shown to play a significant role in the pathology of autoimmune diseases. For example, the presence of IFN-gamma has been implicated in rheumatoid arthritis (Brennan et al, Brit. J. Rheum., 31, 293-8 (1992)). Several strategies to antagonize the action of these cytokines have been developed and are currently used to treat various disease states.
  • IFN-gamma in its bioactive form is a dimer and the groove formed by the two subunits is important for its biological activity through interaction with the IFN-gamma receptor. An IFN-gamma inhibitor which has sufficient specificity and selectivity to IFN-gamma may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory disorders. Diseases associated with IFN-gamma include multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, and psoriatic arthritis (U.S. Pat. No. 6,333,032 Advanced Biotherapy Concepts, Inc.). Other diseases include Crohn's disease and psoriasis (U.S. Pat. No. 6,329,511 Protein Design Labs). Yet other diseases are bowel disease, ulcerative colitis and Crohn's disease (EP0695189 Genentech).
  • None of the presently available drugs are completely effective for the treatment of autoimmune disease, and most are limited by severe toxicity. In addition, it is extremely difficult and a lengthy process to develop a new chemical entity (NCE) with sufficient potency and selectivity to such target sequence. Antibody-based therapeutics on the other hand have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. In addition, the development time can be reduced considerably when compared to the development of new chemical entities (NCE's). However, conventional antibodies are difficult to raise against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with IFN-gamma.
  • The heterospecific polypeptide constructs of the present invention, wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties thereof such as stability, size, and reliable expression. Furthermore, the inventors have found that, despite presence of a groove in multimeric IFN-gamma, the heterospecific polypeptide constructs are still able to achieve strong binding to IF NA-gamma.
  • According to one aspect of the invention, a target against which one or more anti-target single domain antibodies of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is interferon-gamma (IFN-gamma). IFN-gamma is secreted by some T cells. In addition to its anti-viral activity, IFN-gamma stimulates natural killer (NK) cells and T helper 1 (Th1 ) cells, and activates macrophages and stimulates the expression of MHC molecules on the surface of cells. Hence, IFN-gamma generally serves to enhance many aspects of immune function, and is a candidate for treatment of disorders where the immune system is over-active e.g. Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-lFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 25 to 27. The anti-IFN-gamma single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IFN-gamma.
  • Anti-target single domain antibodies may be directed against whole IFN-gamma or a fragment thereof, or a fragment of a homologous sequence thereof.
  • One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of the disorders wherein the immune system is overactive, as mentioned above. Current therapy consists of intravenous administration of anti-IFN-gamma antibodies. Oral delivery of these heterospecific polypeptide constructs results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain IFN-gamma producing cells. These heterospecific polypeptide constructs can neutralise the IFN-gamma locally, avoiding distribution throughout the whole body and thus limiting negative side-effects. Genetically modified microorganisms such as Micrococcus lactis are able to secrete antibody fragments. Such modified microorganisms can be used as vehicles for local production and delivery of antibody fragments in the intestine. By using a strain which produces a IFN-gamma neutralising heterospecific polypeptide construct, inflammatory bowel disorder could be treated.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is over active, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a method of treating, preventing and/or alleviating disorders wherein the immune system is overactive, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.
  • Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies joined to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive.
  • Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is over active.
  • It is an aspect of the invention that the anti-IFN-gamma single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.
  • The above aspect and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IFN-gamma VHHs fused to one or more anti-serum protein single domain antibodies wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 25 to 27, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion. SEQ ID NOS: 25 to 27 comprise anti-IFN-gamma VHH and anti-mouse serum albumin VHH.
  • The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein VHHs wherein said anti-serum protein VHHs correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.
  • One embodiment of the present invention is a recombinant done comprising nucleic acid encoding a heterospeclfic polypeptide construct according to the invention. In one aspect of the invention, said nucleic acid encodes one or more single domain antibodies each directed to a therapeutic or diagnostic target antigen and one or more single domain antibodies directed to a serum protein, said single domain antibodies linked without intervening linkers, or with one or more peptide linker sequences. According to one aspect of the invention, a linker sequence is any suitable linker sequence known in the art. According to another aspect of the invention, a linker sequence is a naturally occurring sequence. Preferred properties of linkers sequences are that they are not immunogenic or not significantly immunogenic, they can provide sufficient flexibility to the heterospecific polypeptide construct, and are resistant to proteolytic degradation. An example of a linker according to the invention is that disclosed in PCT/EP96/01725 which is derived from the hinge region of VHH.
  • According to another aspect of the invention, a done comprises nucleic acid encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion, and nucleic acid encoding one or more anti-target single domain antibodies, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • According to another aspect of the invention, a clone comprises nucleic acid capable of encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS:5 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.
  • It is within the scope of the invention that nucleic acid encoding multiple anti-target and/or multiple anti-serum VHHs are present in a clone of the invention.
  • By transforming a compatible host with a clone encoding a heterospecific polypeptide construct of the invention, the heterospecific polypeptide construct can be produced in sufficient quantities for use in therapy. Examples of organisms into which said clone may be transformed include, but are not limited to E. coli or Sacchoromyces cerevisiae.
  • Another embodiment of the present invention is a method for prolonging the half-life of an anti-target-VHH comprising the step of joining thereto one or more anti-serum albumin single domain antibodies. As already mentioned above, methods for joining are known in the art or may be any future method, for example, they may be fused by chemical coupling, fused at the DNA level etc.
  • Treating, preventing and/or alleviating the symptoms of one or more of the disorders mentioned herein generally involves administering to a subject a “therapeutically effective amount” of heterospecific polypeptide construct. By “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” means the amount needed to achieve the desired result or results. One of ordinary skill in the art will recognise that the potency and, therefore, an “effective amount” can vary for the various compounds that inhibit a disorder pathway used in the invention. One skilled in the art can readily assess the potency of the compound.
  • As used herein, the term “compound” refers to a heterospecific polypeptide construct as disclosed herein, a polypeptide represented by SEQ ID NOS: 5 to 27, a homologous sequence thereof, or a homologue thereof, or a nucleic acid capable of encoding said polypeptide.
  • By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • The Invention disclosed herein is useful for treating or preventing a condition relating to a disorder as mentioned herein (e.g. allergy and/or inflammation), in a subject and comprising administering a pharmaceutically effective amount of a compound or composition that binds to a component involved in the disorder pathway (e.g. to IgE and/or TNF-alpha in the blood stream), so inhibiting the disorder pathway and the disorder.
  • One aspect of the present invention is the use of compounds of the invention for treating or preventing a condition relating to a disorder as mentioned herein (e.g. allergy and/or inflammation), in a subject and comprising administering a pharmaceutically effective amount of a compound in combination with another, such as, for example, aspirin.
  • The present invention is not limited to the administration of formulations comprising a single compound of the invention. It is within the scope of the invention to provide combination treatments wherein a formulation is administered to a patient in need thereof that comprises more than one compound of the invention.
  • It is well known in the art how to determine the inhibition of a disorder pathway using the standard tests described herein, or using other similar tests. Preferably, the method would result in at least a 10% reduction in an indicator of the disorder, including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably by 90%. For example, an inhibition of an allergic pathway by inhibition of IgE by a peptide of the invention might result in a 10% reduction in food-specific IgE levels.
  • The compound useful in the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or any animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intranasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes.
  • The compound of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene for the compound of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells.
  • Thus, the present compound may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
  • The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • For topical administration, the present compound may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the present compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositons which can be used to deliver the compound to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • Useful dosages of the compound can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • Generally, the concentration of the compound(s) in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the compound varies depending on the target cell, tumor, tissue, graft, or organ.
  • The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • An administration regimen could include long-term, daily treatment. By “long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill In the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage can also be adjusted by the individual physician in the event of any complication.
    • EXAMPLES Example 1 Immunization of Llamas
  • One llama was Immunized with human serum albumin (HSA). The immunization scheme is summarized in Table 1.
    • Example 2 Repertoire Cloning
  • Peripheral blood lymphocytes (PBLs) were isolated by centrifugation on a density gradient (Ficoll-Paque Plus Amersham Biosciences). PBLs were used to exitact total RNA (Chomczynski and Sacchi 1987). cDNA was prepared on 100 μg total RNA with MMLV Reverse Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides. The cDNA was purified with a phenol/chloroform extraction, followed by an ethanol precipitation and subsequently used as template to amplify the VHH repertoire.
  • In a first PCR, the repertoire of both conventional (1.6 kb) and heavy-chain (1.3 kb) antibody gene segments were amplified using a leader specific primer (5′-GGCTGAGCTCGGTGGTCCTGGCT-3′) (SEQ ID No 41) and the oligo d(T) primer (5′-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3′) (SEQ ID No 42). The resulting DNA fragments were separated by agarose gel electrophoresis and the 1.3 kb fragment, encoding heavy-chain antibody segments was purified from the agarose gel. A second PCR was performed using a mixture of FR1 reverse primers and the same oligo d(T) forward primer. The PCR products were digested with Sfil (introduced in the FR1 primer) and BstEll (naturally occurring in FR4). Following gel electrophoresis, the DNA fragment of approximately 400 basepairs were purified from gel and ligated into the corresponding restriction sites of phagemid pAX004 to obtain a library of cloned VHHs after electroporation of Escherichia coli TG1. The size of the library was 1.4×107 cfu, and all clones contained insert of the correct size.
    • Example 3 Rescue of the Library, Phage Preparation
  • The library was grown at 37° C. in 10 ml 2×TY medium containing 2% glucose, and 100 μg/ml ampicillin, until the OD600 nm reached 0.5. M13KO7 phages (1012) were added and the mixture was incubated at 37° C. for 2×30 minutes, first without shaking, then with shaking at 100 rpm. Cells were centrifuged for 10 minutes at 4500 rpm at room temperature. The bacterial pellet was resuspended in 50 ml of 2×TY medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, and incubated overnight at 37° C. with vigorously shaking at 250 rpm. The overnight cultures were centrifuged for 15 minutes at 10,000 rpm at 4° C. Phages were PEG precipitated (20% poly-ethylene-glycol and 1.5 M NaCl) and centrifuged for 30 minutes at 10,000 rpm. The pellet was resuspended in 20 ml PBS. Phages were again PEG precipitated and centrifuged for 30 minutes at 20,000 rpm and 4° C. The pellet was dissolved in 5 ml PBS-1% casein. Phages were titrated by infection of TG1 cells at OD600 nm=0.5 and plating on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. The number of transformants indicates the number of phages (=pfu). The phages were stored at −80° C. with 15% glycerol.
    • Example 4 Phage ELISA
  • A microtiter plate (Maxisorp) was coated overnight at 4° C. with PBS-1% casein or with 5 μg/ml HSA (human serum albumin). The plate was washed 3 times with PBS-Tween (0.05% Tween20) and blocked for 2 hours at room temperature with 200 μl PBS-1% casein. The plate was washed five times with PBS-Tween. Phages were prepared as described above and applied to the wells in consecutive twofold dilutions. Plates were washed five times with PBS-Tween. Bound phage were detected with a mouse monoclonal antibody anti-M13 conjugated with horse radish peroxidase (HRP) diluted 1/2000 in PBS. The plates were washed five times with PBS-Tween. Staining was performed with ABTS/H2O2 and signals were measured after 30 minutes at 405 nm. Results are shown in FIG. 1 and indicate the presence of HSA-specific nanobodies in the library.
    • Example 5 Selection: First and Second Round of Biopanning
  • A well in a microtiterplate was coated with 10 μg/ml mouse serum albumin (MSA), or with PBS containing 1% casein. After overnight incubation at 4° C., the wells were blocked with PBS containing 1 % casein, for 3 hours at room temperature (RT). 200 μl phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10× with PBS-Tween and 10× with PBS. Bound phages were eluted with 100 μl 0.2 M glycin buffer pH=2.4. Elutions were performed for 20 minutes at room temperature. Eluted phages were allowed to infect exponentially growing E.Coli TG1 cells, and were then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. A second round was performed with the same conditions as described above. Results are summarized in Table 2.
    • Example 6 Screening of Individual Clones After Biopanning
  • ELISA: binding to human serum albumin (HSA) and mouse serum albumin (MSA)
  • A single colony was used to start an overnight culture in LB containing 2% glucose and 100 μg/ml ampicillin. This overnight culture was diluted 100-fold in TB medium containing 100 μg/ml ampicillin, and incubated at 37° C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37° C. or overnight at 28° C. Cultures were centrifuged for 20 minutes at 10,000 rpm at 4° C. The pellet was frozen overnight or for 1 hour at −20° C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4° C. at 20,000 rpm. The supernatant containing the VHH was used for further analysis.
  • A microtiter plate was coated with 5 μg/ml HSA, with 5 μ/ml mouse serum albumin (MSA) or with PBS-1% casein, overnight at 4° C. Plates were blocked for two hours at room temperature with 300 μl 1% casein in PBS. The plates were washed three times with PBS-Tween. Periplasmic fraction was prepared for 23 individual clones after the first and second round of selection, and allowed to bind to the wells of the microtiterplate. Plates were washed six times with PBS-Tween, after which binding of nanobody was detected by incubation with mouse anti-Histidine monoclonal antibody Serotec MCA 1396 ( 1/1000 dilution) in PBS for 1 hour at RT followed by anti-mouse-alkaline phosphatase conjugate 1/2000 in PBS, also for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg2SO4, pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are summarized in Table 3.
    • Example 7 Hinfl Pattern and Sequencing
  • A PCR was performed on positive clones after the second round of panning, with a set of primers binding to a sequence in the vector. The PCR product was digested with the restriction enzyme Hinfl and loaded on a agarose gel. 4 clones were selected with a different Hinfl-pattern for further evaluation. Those clones were sequenced, and results are summarized in Table 4 (SEQ ID NOS: 1, 2 ,3 and 4).
    • Example 8 Test Cross-reactivity with Albumin of Different Species
  • A SDS-PAGE was run for plasma ( 1/10 dilution) from different species (baboon, pig, hamster, human, rat, mouse and rabbit) and blotted on a nitrocellulose membrane. Phages were prepared for clones MSA 21, MSA 24, MSA 210, MSA212 and a control nanobody as described in Example 3. Phages were allowed to bind to the nitrocellulose blotted serum albumins and unbound phages were washed away. Binding was detected with an anti-M13 polyclonal antibody coupled to HRP. DAP was used as a substrate for detection. Results are shown in FIG. 2.
  • From these results we can conclude that all 4 binders are cross-reactive between pig, human, mouse (less for MSA212) and hamster serum albumin. MSA 21 is also cross-reactive with rabbit serum albumin. With the irrelevant nanobody no binding was observed (not shown).
  • As a control experiment, a SDS-PAGE was run with the different plasma samples diluted 1/100 in PBS. The gel was stained with coomassie. We can conclude from FIG. 3 that albumin levels in all plasma samples are high except for rabbit plasma, with low levels of albumin.
    • Example 9 Expression and Purification
  • Plasmid was prepared for the binders and was transformed into WK6 electrocompetent cells. A single colony was used to start an overnight culture in LB containing 2% glucose and 100 μg/ml ampicillin. This overnight culture was diluted 100-fold in 300 ml TB medium containing 100 μg/ml ampicillin, and incubated at 37° C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37° C. or overnight at 28° C. Cultures were centrifuged for 20 minutes at 10,000 rpm at 4° C. The pellet was frozen overnight or for 1 hour at −20° C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4° C. at 20,000 rpm. The supernatant containing the nanobody was loaded on Ni-NTA and purified to homogeneity.
    • Example 10 ELISA on MSA of the Purified Nanobodies
  • A microtiterplate was coated with 5 μg/ml MSA overnight at 4 C. After washing, the plate was blocked for 2 hours at RT with PBS-1% casein. Samples were applied in duplicate starting at a concentration of 2500 nM at ⅓ dilutions and allowed to bind for 2 hours at RT. A polyclonal rabbit anti-nanobody serum was added at 1/1000 (K208) for one hour at RT. Detection was with ant-rabbit alkaline phosphatase conjugate at 1/1000 and staining with PNPP as described in Example 6. Results are shown in FIG. 4.
    • Example 11 Construction of Bispecific Constructs
  • The E. coli production vector pAX11 was constructed to allow the two-step cloning of bivalent or bispecific VHH (FIG. 5).
  • The carboxy terminal VHH was cloned first with Pstl and BstEll, while in the second step the other VHH was inserted by Sfil and Notl, which do not cut within the first gene fragment. The procedure avoids the enforcement of new sites by amplification and thus the risk of introducing PCR errors. The middle hinge of llama was used as a linker between the nanobodies. A VHH against human TNF alpha was cloned at the COOH terminal of MSA specific nanobodies. Sequences are summarized in Table 4 (SEQ ID NOS: 5, 6, 7 and 8). Plasmid was prepared and was transformed into WK6 electrocompetent cells. A single colony was used to start an overnight culture in LB containing 2% glucose and 100 μg/ml ampicillin. This overnight culture was diluted 100-fold in 300 μl TB medium containing 100 mg/ml ampicillin, and incubated at 37° C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37° C.
  • Cultures were centrifuged for 20 minutes at 10,000 rpm at 4° C. The pellet was frozen overnight at −20 C. The next morning, the pellet was thawed in the cold room for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4° C. at 10,000 rpm. The supernatant was loaded on Ni-NTA and purified to homogeneity. Sequences are shown in Table 4 (SEQ ID NOS: 5, 6, 7 and 8). A extra purification step was needed to remove some degradation product (5%) on gelfiltration.
  • Another bispecific VHH against human TNF-alpha (MP7 12 b) is listed in Table 4 (SEQ ID NOS: 15, 16, 17 and 18).
    • Example 12 Test Bispecific Construct in Sandwich ELISA
  • A microtiter plate was coated with 5 μg/ml MSA overnight at 4° C. Plates were blocked for two hours at room temperature with 300 μl 1% casein in PBS. The plates were washed three times with PBS-Tween. Purified protein for the bispecific constructs was allowed to bind to the wells of the microtiterplate at a concentration of 0.4, 0.5, 2.5 and 2.5 μg/ml for MSA21, MSA24, MSA210 and MSA212 respectively. Plates were washed six times with PBS-Tween, Biotinilated TNF was added at a concentration of 10 μg/ml and diluted 3 fold, and allowed to bind for 2 hours at room temperature. Binding was detected by incubation with mouse extravidin alkaline phosphatase conjugate (Sigma) 1/2000 in PBS, for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg2SO4, pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are shown in FIG. 6 and indicate that the bispecific construct can bind both antigens simultaneously.
    • Example 13 Determine Affinity of Albumin Binders In BIACORE
  • Affinities for mouse albumin were determined in BIACORE by immobilization of mouse albumin on a CM5 BIAcore chip using EDC-NHS covalent coupling and are summarized in Table 5. The results indicate that the affinity for albumin is retained in the bispecific construct.
    • Example 14 Optimization of ELISA in Plasma or Blood
  • Pharamcokinetic experiments were initiated to compare half life in mice of the TNF-alpha binder TNF3E with MSA21/VHH#3E and MSA24/VHH#3E. Therefore our ELISA had to be optimized to obtain low background values when the samples are in blood or in plasma. A microtiterplate was coated with neutravidin. After overnight incubation at 4 C, the plates were washed and blocked for 2 hours at RT with PBS-1% casein. 1 μg/ml biotinylated TNF-alpha was allowed to bind for 30 minutes at RT and the plate was washed. Samples (monovalent VHH#3E and MSA21/VHH#3E) were applied starting at a concentration of 1 μg/ml, diluted in PBS, 10% plasma or 10% blood and allowed to bind for 2 hours. After washing the plates, a rabbit antiserum was added at a dilution of 1/2000 either recognizing the heavy chain class (K208) or recognizing the conventional class (URL49). After 1 hour incubation, the plates were washed and an anti-rabbit alkaline phosphatase conjugate was added (Sigma) at a dilution of 1/1000. After 1 hour incubation at RT, plates were washed and binding was detected with substrate. Results are shown in FIG. 7. The results clearly show that background values with the rabbit antisera (K208 and URL49) are very low when the samples are diluted in 10% blood or 10% plasma as compared to PBS. The URL49 antiserum only recognizes the MSA21/VHH#3E bispecific nanobody and not monovalent VHH#3E, therefore, this antiserum can be used to test the integrity of our bispecific nanobody upon administration to the mice.
    • Example 15 Large Scale Expression and Purification of VHH#3E, MSA21/VHH#3E and MSA24/VHH#3E for Pharmacokinetic Studies in Mice
  • 3 liter culture was started for monovalent TNF3E and for bispecific MSA21/VHH#3E or MSA24/VHH#3E and purified as described in Example 11. An extra purification step was needed for the removal of endotoxins. Therefore, samples were purified on a Polymyxin column (BIO-RAD). Samples were analyzed for bacterial endotoxin concentration with the LAL-assay (Limulus Amebocyte Lysate, Bio Whittaker). Results are summarized in Table 6.
    • Example 16 Pharmacokinetics in Mice
  • 9 mice (CB57/BI6) for each construct were injected intravenously in the tail with 100 μg nanobody. Blood was retrieved at different time points (3 mice per time point) and serum was prepared. Samples were analyzed by ELISA for the presence of monovalent or bispecific nanobody as described in example 14. K208 was also compared to URL49 for the bispecific constructs to verify the integrity of the molecule. Results are shown in FIGS. 8 to 11.
  • We can conclude from the results that the half life of the monovalent nanobody (40-45 minutes) is dramatically improved by making a bispecific nanobody with specificity for albumin MSA21/VHH#3E and MSA24/VHH#3E (half-life 2.5 to 3 days). The bispecific nanobody MSA21/VHH#3E remains intact even after 19 days in the mice as shown in ELISA with URL49 (FIG. 11).
    • Example 17 Further Extension of Half-life of Nanobodies
  • In order to increase the half-life of MSA21/TNF3E and MSA24/TNF3E even further, a trivalent nanobody was prepared by fusing the bivalent MSA21-MSA21 construct to target-specific nanobody TNF3E. The resulting MSA21/MSA21/TNF3E (Table 7, and SEQ ID NO: 9) was tested in vivo according to the method of Example 16.
    • Example 18 Immunization of Llama002
  • 1 llama was immunized with vWF. The immunization scheme is summarized in Table 7.
    • Example 19 Repertoire Cloning and Phage Preparation
  • The library was prepared as described in Example 2. The size of the library was 1.4×107 cfu, and >90% of the clones contained insert of the correct size. Phages were prepared as described in Example 3.
    • Example 20 Selection for Binders for vWF Inhibiting the Interaction with Collagen: First and Second Round of Panning
  • A well in a microtiterplate was coated with 2 μg/ml vWF or with PBS containing 1% casein. After overnight incubation at 4° C., the wells were blocked with PBS containing 1% casein, for 3 hours at RT. 200 μl phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10× with PBS-Tween and 10× with PBS. Phages were specifically eluted with 100 μl of 100 μg/ml collagen type III. Elutions were performed for overnight at room temperature. Eluted phages were allowed to infect exponentially growing TG1 cells, and were then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. This experiment was repeated for a second round of panning, under the same conditions as described above. The results from the panning are presented in Tables 8 and 9.
    • Example 21 Functional Characterization of vWF Binders: Inhibition of Binding of vWF to Collagen by VHH
  • A microtiter plate was coated overnight at 4° C. with collagen type III at 25 μg/ml in PBS. The plate was washed five times with PBS-Tween and blocked for 2 hours at room temperature with PBS containing 1% casein. The plate was washed five times with PBS-tween. 100 μl of 2 μg/ml vWF (vWF is pre-incubated at 37° C. for 15 minutes) was mixed with 20 μl periplasmic extract containing a VHH antibody (described in Example 6) and incubated for 90 minutes at room temperature in the wells of the microtiterplate. The plate was washed five times with PBS-tween. An anti-vWF-HRP monoclonal antibody (DAKO) was diluted 3,000-fold in PBS and incubated for 1 hour. The plate was washed five times with PBS-Tween and vWF-binding was detected with ABTS/H2O2. Signals were measured after 30 minutes at 405 nm. The results are presented in Table 10, showing that inhibitors are obtained after the first and second round of panning.
    • Example 22 Expression and Purification of VHH
  • Protein was prepared and purified as described in Example 9.
    • Example 23 ELISA: Binding to vWF
  • A microtiter plate was coated with 2 μg/ml vWF, overnight at 4° C. Plates were blocked for two hours at room temperature with 300 μl 1% casein in PBS. The plates were washed three times with PBS-Tween. Dilution series of all purified samples were incubated for 2 hours at RT. Plates were washed six times with PBS-Tween, after which binding of VHH was detected by incubation with mouse anti-myc mAB 1/2000 in PBS for 1 hour at RT followed by anti-mouse-HRP conjugate 1/1000 in PBS, also for 1 hour at RT. Staining was performed with the substrate ABTS/H2O2 and the signals were measured after 30 minutes at 405 nm. The binding as a function of concentration of purified VHH is indicated in FIG. 12.
    • Example 24 Inhibition ELISA with Purified VHH
  • Inhibition ELISA was performed as described in Example 20 but with decreasing concentrations of VHH and with human plasma at a dilution of 1/60 instead of with purified vWF. Results are represented in FIG. 13. The concentration of VHH resulting in 50% inhibition (IC50) is given in table 10.
    • Example 25 Construction and Sequence of Bispecific Constructs
  • Bispecific constructs were prepared with the first VHH specific for albumin (MSA21) and the second VHH specific for vWF. Constructs were made as described in Example 11. Sequences are shown in Table 4 (SEQ ID NOS: 19 to 21)
    • Example 26 Expression and Purification of Bispecific Constructs
  • Protein was expressed and purified as described in Example 9. An extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%).
    • Example 27 Functionality of Both VHHs in the Bispecific Construct
  • A microtiterplate was coated with 5 μg/ml mouse serum albumin overnight at 4° C. After washing the plate, wells were blocked for 2 hours with PBS-1% casein. The bispecific proteins were allowed to bind to the wells for 2 hours at RT. After washing, human, dog and pig plasma was added at different dilutions and allowed to bind for 2 hours at RT. Binding of vWF was detected with anti-vWF-HRP from DAKO at 1/3000 dilution. Staining was performed with ABTS/H2O2. Results are shown in FIG. 14 and indicate that functionality of both VHHs is retained in the bispecific construct.
    • Example 28 Inhibition of Binding of vWF to Collagen by the Bispecific Constructs as Compared to the Monovalent VHHs
  • Inhibition for binding of vWF to collagen was tested for monovalent as compared to bispecific constructs as described in Example 20. IC50 values are summarized in Table 11. Results indicate that the inhibitory properties of the VHH are retained in the bispecific construct.
    • Example 29 Construction of a Bispecific Construct Containing a VHH-CDR3 Fragment Fused to an Anti-serum Albumin VHH
  • A functional portion, the CDR3 region of MP2F6SR, was amplified by using a sense primer located in the framework 4 region (F6 CRD3 Forward:CTGGCCCCAGAAGTCATACC) (SEQ ID No 43) and an anti-sense primer located in the framework 3 region (F6 CDR3 Reverse primer:TGTGCATGTGCAGCAAACC) (SEQ ID No 44).
  • In order to fuse the CDR-3 fragment with the anti-serum albumin VHH MSA-21, a second round PCR amplification was performed with following primers:
    F6 CDR3 Reverse primer Sfi1:
    (SEQ ID N° 45)
    GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCATGTGCAGCAAACC
    F6 CDR3 Forward primer Not1:
    (SEQ ID N° 46)
    GTCCTCGCAACTGCGCGGCCGCCTGGCCCCAGAAGTCATACC
  • The PCR reactions was performed in 50 ml reaction volume using 50 pmol of each primer. The reaction conditions for the primary PCR were 11 min at 94° C., followed by 30/60/120 sec at 94/55/72° C. for 30 cycles, and 5 min at 72° C. All reaction were performed wit 2.5 mM MgCl2 , 200 mM dNTP and 1.25 U AmpliTaq God DNA Polymerase (Roche Diagnostics, Brussels, Belgium).
  • After cleavage of the VHH gene of MSA clones with restriction enzymes Pst1/BstEll the digested products were cloned in pAX11 to obtain clones with a VHH at the C-terminus of the multicloning site. The clones were examined by PCR using vector based primers. From clones yielding a 650 bp product, DNA was prepared and used as acceptor vector to clone the CDR3 of MP2F6SR after cleavage of the PCR product with restriction enzymes Sfi1/Not1 to allow N-terminal expression of CDR3 in fusion with a MSA VHH.
    • Example 30 Calculation of Homologies Between Anti-target Single Domain Antibodies of the Invention
  • The degree of amino acid sequence homology between anti-target single domain antibodies of the invention was calculated using the Bioedit Sequence Alignment Editor. The calculations indicate the proportion of identical residues between all of the sequences as they are aligned by ClustalW. (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research, submitted, June 1994). Table 12 indicates the fraction homology between anti-serum albumin VHHs of the invention. Table 13 indicates the fraction homology between anti-TNF-alpha VHHs of the invention. Table 14 indicates the percentage homology between anti-IFN-gamma VHHs of the invention. Table 15 indicates the fraction homology between anti-vWF VHHs of the invention.
    TABLE 1
    Immunization scheme according to Example 1
    HSA
    Day of immunization Llama006
    0 100 μg 
    7 100 μg 
    14 50 μg
    21 50 μg
    28 50 μg
    35 50 μg
  • TABLE 2
    results after one and two rounds of panning on mouse serum albumin as
    described in example 5.
    First round Second round
    Pfu mouse serum albumin 2.5 × 107 2.5 × 107
    Pfu casein   5 × 103 2.5 × 103
    enrichment 5,000 10,000
  • TABLE 3
    Clones were selected after one and two rounds of selection and
    peripiasmic extracts were prepared. These clones were analyzed
    in ELISA for binding to human and mouse albumin as described
    in Example 6.
    First round Second round
    ELISA mouse serum albumin 1/16 15/16
    ELISA human serum albumin 1/16 15/16
    ELISA casein 0/16  0/16
  • TABLE 4
    Sequence listing
    NAME SEQ ID SEQUENCE
    Anti-mouse serum albumin
    MSA21 1 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSS
    MSA24 2 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW
    VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR
    DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG
    TQVTVSS
    MSA210 3 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSS
    MSA212 4 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW
    VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR
    DNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPASQGT
    QVTVSS
    MSAc16 28 AVQLVESGGGLVQAGDSLRLSCVVSGTTFSSAAMGW
    FRQAPGKEREFVGAIKWSGTSTYYTDSVKGRFTISR
    DNVKTVYLQMNNLKPEDTGVYTCAADRDRYRDRMGP
    MTTTDFRFWGQGTQVTVSS
    MSAcl12 29 QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGW
    FRQAPGREREFVASIGSSGITTNYADSVKGRFTISR
    DNAKNTVYLQMNSLKPEDTGLCYCAVNRYGIPYRSG
    TQYQNWGQGTQVTVSS
    MSAcl10 30 EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGW
    YRQAPGKERDMVATISIGGRTYYADSVKGRFTISRD
    NAKNTVYLQMNSLKPEDTAIYYCVAHRQTVVRGPYL
    LWGQGTQVTVSS
    MSAcl14 31 QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGW
    FRQAPGKEREFVAGSGRSNSYNYYSDSVKGRFTISR
    DNAKNTVYLQMNSLKPEDTAVYYCAASTNLWPRDRN
    LYAYWGQGTQVTVSS
    MSAcl16 32 EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMGW
    FRQVPGKEREFVAAISWSGGTTRYIDSVKGRFTISR
    DSTKNAVYLQMNSLKPEDTAVYYCAVDSSGRLYWTL
    STSYDYWGQGTQVTVSS
    MSAcl19 33 QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIYKMAW
    FRQVPGKEREFVAAISWSGGTTRYIDSVKGRFTLSR
    DNTKNMVYLQDMNSLKPDDTAVYYCAVDSSGRLYWT
    LSTSYDYWGQGTQVTVSS
    MSAc15 34 EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGW
    FRQAPGKEREFLAGVTWSGSSTFYGDSVKGRFTASR
    DSAKNTVTLEMNSLNPEDTAVYYCAAAYGGGLYRDP
    RSYDYWGRGTQVTVSS
    MScl11 35 AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAW
    FRQAPGKEREGVSCIRDGTTYYADSVKGRFTISSDN
    ANNTVYLQTNSLKPEDTAVYYCAAPSGPATGSSHTF
    GIYWNLRDDYDNWGQGTQVTVSS
    MSAcl15 36 EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGW
    FRQVPGKEREGVSCISSSDGSTYYADSVKGRFTISS
    DNAKNTVYLQMNTLEPDDTAVYYCAAGGLLLRVEEL
    QASDYDYWGQGIQVTVSS
    MSAcl8 37 AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGW
    FRQAPGKEREGVACISNSDGSTYYGDSVKGRFTISR
    DNAKTTVYLQMNSLKPEDTAVYYCATADRHYSASHH
    PFADFAFNSWGQGTQVTVSS
    MSAcl7 38 EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMAW
    FRRAPGKERELVVARNWGDGSTRYADSVKGRFTISR
    DNAKNTVYLQMNSLKPEDTAVYYCAAVRTYGSATYD
    IWGQGTQVTVSS
    MSAcl20 39 EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYAMGW
    FRQAPGKEREFVAAINRNGGTTNYADALKGRFTISR
    DNTKNTAFLQMNSLKPDDTAVYYCAAREWPFSTIPS
    GWRYWGQGTQVTVSS
    MSAcl4 40 DVQLVESGGGWVQPGGSLRLSCAASGPTASSHAIGW
    FRQAPGKEREFVVGINRGGVTRDYADSVKGRFAVSR
    DNVKNTVYLQMNRLKPEDSAIYICAARPEYSFTAMS
    KGDMDYWGKGTLVTVSS
    Anti-mouse serum albumin/anti TNF-alpha
    MSA21/ 5 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    VHH#3E VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
    LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFV
    ARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMN
    NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV
    TVSS
    MSA24/ 6 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW
    VHH#3E VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR
    DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
    LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFV
    ARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMN
    NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV
    TVSS
    MSA210/ 7 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#3E VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA
    RIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNN
    LEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVT
    VSS
    MSA212/ 8 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW
    VHH#3E VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR
    DNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPASQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA
    RIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNN
    LEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVT
    VSS
    MSA21/ 9 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    MSA21/ VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    VHH#3E DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
    LRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISS
    LGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPE
    DTAVYYCTIGGSLNPGGQGTQVTVSSEPKTPKPQPA
    AAQVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSG
    YTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVK
    GRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDG
    IPTSRSVESYNYWGQGTQVTVSS
    MSA210/ 10 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#1 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCATSGFDFSVSWMYWVRQAPGKGLEWVSEINTN
    GLITKYVDSVKGRFTISRDNAKNTLYLQMDSLIPED
    TALYYCARSPSGSFRGQGTQVTVSS
    MSA210/ 11 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#9 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGSIFRVNAMGWYRQVPGNQREFVAIITSG
    DNLNYADAVKGRFTISTDNVKKTVYLQMNVLKPEDT
    AVYYCNAILQTSRWSIPSNYWGQGTQVTVSS
    MSA210/ 12 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#13 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCATSGFTFSDYWMYWVRQAPGKGLEWVSTVNTN
    GLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSED
    TAVYYCTKVVPPYSDDSRTNADWGQGTQVTVSS
    MSA210/ 13 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH+1902 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA
    RIYWSSGNTYYADSVKGRFAISRDIAKINTVDLTMN
    NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV
    TVSS
    MSA210/ 14 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#3 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGSL
    RLSCAVSGRTFSAHSVYTMGWFRQAPGKEREFVARI
    YWSSANTYYADSVKGRFTISRDNAKNTVDLLMNSLK
    PEDTAVYYCAARDGIPTSRTVGSYNYWGQGTQVTVS
    S
    MSA21/ 15 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    VHH#12B VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
    LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNT
    NGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSE
    DTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS
    MSA24/ 16 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW
    VHH#12B VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR
    DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
    LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNT
    NGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSE
    DTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS
    MSA210/ 17 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    VHH#12B VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTN
    GLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSED
    TAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS
    MSA212/ 18 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW
    VHH#12B VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR
    GTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGG
    SLRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVN
    TNGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKS
    EDTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS
    Anti-mouse serum albumin/anti-vWF
    MSA21/ 19 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    AM-2-75 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD
    NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
    RLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTY
    GEPRYADSVKADSPSSETTPTTRCICNEQPETEDTA
    VYYCARGAGTSSYLPQRGNWDQGTQVTVSS
    MSA21/ 20 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    AM-4-15- VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD
    3 NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT
    QVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGSL
    RLACAASGSIFSINSMGWYRQAPGKQRELVAHALAD
    GSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDT
    AVYYCNTVPSSVTKGYWGQGTQVTVSS
    MSA21/ 21 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    22-4L-16 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD
    NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT
    QVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSL
    RLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS
    GGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED
    TAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS
    Anti-mouse serum albumin/anti-IgE
    MSA21/ 22 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    EV 2H11 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGS
    LRLSCAASGVTFSSYAMGWFRQAPGKEREFVASITW
    TGTGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPE
    DTAVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS
    MSA 24/ 23 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW
    EV 2H11 VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR
    DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG
    TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGS
    LRLSCAASGVTFSSYAMGWFRQAPGKEREFVASITW
    TGTGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPE
    DTAVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS
    MSA 210/ 24 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    EV 2H11 VRQAPGKGLEWVSAISSDSGTKYADSVKGRFTISRD
    NAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGTQ
    VTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSLR
    LSCAASGVTFSSYAMGWFRQAPGKEREFVASITWTG
    TGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPEDT
    AVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS
    Anti-mouse serum albumin/anti-IFN-gamma
    MSA21/ 25 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW
    MP2F6SR VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR
    DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG
    TQVTVSSEPKTPKPQPAAAQVKLEESGGGLVQAGGS
    LRLSCAASGRTFNNYNMGWFRQAPGKEREFVAAISW
    NGGSTYYDDSVKGRFTISRDNANNLVYLQMNSLNFE
    DTAVYYCACAAANYGIPQYRENRYDFWGQGTQVTVS
    S
    MSA24/ 26 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW
    MP2F1BR VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR
    DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG
    TQVTVSSEPKTPKPQPAAAAVQLVESGGGLVQTGDS
    LRLSCVASGGTFSRYAMGWFRQAPGKEREFVARIGY
    SGRSISYATSVEGRFAISRDNAKNTVYLQMNSLKPE
    DTAVYYCASLVSGTLYQADYWGQGTQVTVSS
    MSA 210/ 27 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW
    MP3HGSRA VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR
    DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT
    QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSL
    RLSCAASGRTFSIYNMGWFRQAPGKEREFVAGISWN
    GGSIYYTSSVEGRFTISRDNAENTVYLQMNSLKPED
    TGVYYCASKGRPYGVPSPRQGDYDYWGQGTQVTVSS
  • TABLE 5
    Affinities (koff, kon and KD) for albumin binders as determined
    by BIACORE as described in Example 13.
    Kon (105 M−1s−1) Koff (10−5s−1) KD [nM]
    MSA21 3.4 420 12
    MSA24 6.4 1800 28
    MSA212 3.7 9330 250
    MSA21/TNF3E 2.3 370 16
    MSA24/TNF3E 3.1 630 20
    MSA212/TNF3E 0.42 490 120
  • TABLE 6
    Results for the LAL-assay for monovalent and bispecific nanobodies after
    purification on polymyxin as described in Example 15.
    Monovalent Bispecific Bispecific
    TNF3E MSA21/TNF3E MSA24/TNF3E
    Endotoxin units/mg 0.13 Eu/mg 0.75 Eu/mg 2.8 Eu/mg
    of VHH
  • TABLE 7
    Immunization scheme used for llama 002 according to Example 17.
    Llama002
    Day of immunization vWF
    0 100 μg 
    7 100 μg 
    14 50 μg
    21 50 μg
    28 50 μg
    35 50 μg
  • TABLE 8
    Plaque forming units (pfu) after one or two round(s) of panning on vWF as
    compared to PBS-casein as described in example 19. Pfu vWF (antigen)
    divided by pfu casein (a specific binding) = enrichment.
    round Pfu vWF Pfu casein Enrichment
    First
    1 × 107 2.5 × 105 40
    Second 5 × 108 2.5 × 106 200
  • TABLE 9
    Number of Inhibitors versus the number of clones tested after the
    first and the second round of panning as described in Example 20.
    Number of inhibitors versus
    round number of clones tested
    First 4/800
    Second 4/96 
  • TABLE 10
    concentration of VHH (nM) needed to Inhibit binding of vWF to collagen
    by 50% (IC50) as described in Example 23.
    Name VHH IC50 (nM)
    22-2L-34 10
    T76 30
    AM-4-15-3 2
    22-4L-16 0.5
    C37 2
    AM-2-75 2
  • TABLE 11
    IC50 values for bispecific nanobodies against albumin and against vWF as
    described in Example 28.
    IC50 (ng/ml)
    AM-2-75 100
    MSA21/AM-2-75 60
    AM-4-15-3 155
    MSA21/AM-4-15-3 245
    22-4L-16 100
    MSA21/22-4L-16 140
  • TABLE 12
    Fractional homologies between the amino acid sequences of anti-mouse
    serum albumin VHHs of the invention.
    SEQ MSA21 MSA24 MSA210 MSA212
    MSA21 1.000 0.834 0.800 0.782
    MSA24 — 1.000 0.782 0.791
    MSA210 — — 1.000 0.903
    MSA212 — — — 1.000
  • TABLE 13
    Fractional homologies between anti-TNF-alpha VHHs of the invention.
    SEQ VHH#1A VHH#7B VHH#2B VHH#3E VHH#3G VHH#10A VHH#2G VHH#1F
    VHH#1A 1.000 0.601 0.764 0.596 0.622 0.600 0.682 0.629
    VHH#7B — 1.000 0.604 0.635 0.645 0.943 0.653 0.616
    VHH#2B — — 1.000 0.620 0.645 0.611 0.682 0.661
    VHH#3E — — — 1.000 0.875 0.641 0.713 0.689
    VHH#3G — — — — 1.000 0.651 0.779 0.740
    VHH#10A — — — — — 1.000 0.658 0.614
    VHH#2G — — — — — — 1.000 0.741
    VHH#1F — — — — — — — 1.000
    VHH#9C — — — — — — — —
    VHH#11E — — — — — — — —
    VHH#10C — — — — — — — —
    VHH#4B — — — — — — — —
    VHH#10D — — — — — — — —
    VHH#12B — — — — — — — —
    VHH#9E — — — — — — — —
    VHH#3F
    SEQ VHH#9C VHH#11E VHH#10C VHH#4B VHH#10D VHH#12B VHH#9E VHH#3F
    VHH#1A 0.609 0.601 0.614 0.818 0.642 0.747 0.596 0.604
    VHH#7B 0.933 0.933 0.719 0.593 0.614 0.620 0.616 0.324
    VHH#2B 0.629 0.620 0.637 0.796 0.634 0.951 0.620 0.645
    VHH#3E 0.620 0.643 0.612 0.604 0.648 0.596 0.674 0.682
    VHH#3G 0.637 0.637 0.653 0.645 0.689 0.622 0.708 0.716
    VHH#10A 0.935 0.934 0.725 0.592 0.612 0.626 0.622 0.637
    VHH#2G 0.653 0.669 0.685 0.666 0.746 0.650 0.701 0.717
    VHH#1F 0.616 0.616 0.664 0.661 0.714 0.645 0.709 0.717
    VHH#9C 1.000 0.941 0.743 0.601 0.622 0.637 0.608 0.624
    VHH#11E — 1.000 0.719 0.601 0.622 0.637 0.608 0.624
    VHH#10C — — 1.000 0.650 0.606 0.637 0.600 0.632
    VHH#4B — — — 1.000 0.611 0.796 0.588 0.629
    VHH#10D — — — — 1.000 0.619 0.674 0.674
    VHH#12B — — — — — 1.000 0.604 0.637
    VHH#9E — — — — — — 1.000 0.654
    VHH#3F 1.000
  • TABLE 14
    Percentage homologies between anti-IFN-gamma VHHs pf the invention.
    % Homology
    MP3D2SRA MP3A3SR MP3C5SR MP3C1SR MP3G8SR MP3D2BR MP3H6SRA MP3B4SRA MP4E4BR
    MP3D2SRA X 95 66 65 66 82 71 71 71
    MP3A3SR — X 66 66 66 62 72 72 72
    MP3C5SR — — X 97 98 73 65 65 64
    MP3C1SR — — — X 98 72 64 64 64
    MP3G8SR — — — — X 73 65 65 64
    MP3D3BR — — — — — X 63 63 63
    MP3H6SRA — — — — — — X 100 97
    MP3B4SRA — — — — — — — X 97
    MP4E4BR — — — — — — — — X
    MP4H8SR — — — — — — — — —
    MP2F6SR — — — — — — — — —
    MP3D1BR — — — — — — — — —
    MP2B5BR — — — — — — — — —
    MP2C1BR — — — — — — — — —
    MP4A12SR — — — — — — — — —
    MP3F4SRA — — — — — — — — —
    MP3D3BR — — — — — — — — —
    MP3E5BR — — — — — — — — —
    MP3C7SRA — — — — — — — — —
    MP2F1BR — — — — — — — — —
    MP2C5BR — — — — — — — — —
    MP2C10BR — — — — — — — — —
    MP2G5SR — — — — — — — — —
    MP3B1SRA — — — — — — — — —
    MP2F10SR — — — — — — — — —
    MP3A7SRA — — — — — — — — —
    MP4C10SR — — — — — — — — —
    MP4D5BR — — — — — — — — —
    MP3F1SRA — — — — — — — — —
    MP6D6BR — — — — — — — — —
    MP6B1BR — — — — — — — — —
    MP6A8BR — — — — — — — — —
    MP6B12BR — — — — — — — — —
    MP6C11BR
    MP6B10BR
    % Homology
    MP4H8SR MP2F6SR MP3D1BR MP2B5BR MP2C1BR MP4A12SR MP3F4SRA MP3D3SR MP3E5BR
    MP3D2SRA 70 68 69 65 63 64 63 66 67
    MP3A3SR 71 70 71 65 63 64 68 66 67
    MP3C5SR 63 63 63 60 58 59 64 64 65
    MP3C1SR 62 62 62 58 57 58 65 64 64
    MP3G8SR 63 63 63 59 58 59 64 64 65
    MP3D3BR 62 52 64 59 58 58 62 61 62
    MP3H6SRA 97 80 81 67 68 67 75 71 73
    MP3B4SRA 97 80 81 67 68 67 75 71 73
    MP4E4BR 97 81 82 68 69 68 70 70 71
    MP4H8SR X 81 81 66 66 66 72 69 71
    MP2F6SR — X 94 65 68 64 70 67 69
    MP3D1BR — — X 65 66 65 71 69 71
    MP2B5BR — — — X 95 97 63 64 64
    MP2C1BR — — — — X 95 63 64 64
    MP4A12SR — — — — — X 63 64 64
    MP3F4SRA — — — — — — X 64 98
    MP3D3BR — — — — — — — X 98
    MP3E5BR — — — — — — — — X
    MP3C7SRA — — — — — — — — —
    MP2F1BR — — — — — — — — —
    MP2C5BR — — — — — — — — —
    MP2C10BR — — — — — — — — —
    MP2G5SR — — — — — — — — —
    MP3B1SRA — — — — — — — — —
    MP2F10SR — — — — — — — — —
    MP3A7SRA — — — — — — — — —
    MP4C10SR — — — — — — — — —
    MP4D5BR — — — — — — — — —
    MP3F1SRA — — — — — — — — —
    MP6D6BR — — — — — — — — —
    MP6B1BR — — — — — — — — —
    MP6A8BR — — — — — — — — —
    MP6B12BR — — — — — — — — —
    MP6C11BR
    MP6B10BR
    % Homology
    MPC7SRA MP2F1BR MP3C5BR MP3C10BR MP2G5SR MP3B1SRA MP2F10SR MP3A7SRA MP4C10SR
    MP3D2SRA 68 71 70 68 67 63 67 68 60
    MP3A3SR 68 72 72 69 67 64 66 67 60
    MP3C5SR 66 65 65 65 63 63 64 64 61
    MP3C1SR 65 64 63 64 62 63 64 65 60
    MP3G8SR 66 65 64 65 63 63 65 65 61
    MP3D3BR 63 64 63 63 63 64 63 63 63
    MP3H6SRA 75 73 71 73 71 66 75 75 63
    MP3B4SRA 75 73 71 73 71 66 75 75 63
    MP4E4BR 73 73 71 73 71 66 75 75 63
    MP4H8SR 72 71 71 72 71 64 73 73 62
    MP2F6SR 71 67 65 73 71 63 71 70 62
    MP3D1BR 72 67 65 70 69 63 71 71 62
    MP2B5BR 64 65 52 64 52 60 66 63 57
    MP2C1BR 64 63 61 66 65 59 66 63 56
    MP4A12SR 64 62 60 63 62 59 65 52 56
    MP3F4SRA 97 69 67 68 68 62 67 69 60
    MP3D3BR 96 70 68 67 67 62 67 67 60
    MP3E5BR 98 70 68 68 69 63 68 68 60
    MP3C7SRA X 71 69 69 70 63 69 69 61
    MP2F1BR — X 94 66 67 63 68 67 61
    MP2C5BR — — X 66 67 63 67 65 62
    MP2C10BR — — — X 94 62 68 66 59
    MP2G5SR — — — — X 62 67 65 59
    MP3B1SRA — — — — — X 66 65 91
    MP2F10SR — — — — — — X 97 61
    MP3A7SRA — — — — — — — X 61
    MP4C10SR — — — — — — — — X
    MP4D5BR — — — — — — — — —
    MP3F1SRA — — — — — — — — —
    MP6D6BR — — — — — — — — —
    MP6B1BR — — — — — — — — —
    MP6A8BR — — — — — — — — —
    MP6B12BR — — — — — — — — —
    MP6C11BR
    MP6B10BR
    % Homology
    MP4D5BR MP3F1SRA MP6D6BR MP6B1BR MP6A8BR MP6B12BR MP8C11BR MP6B10BR
    MP3D2SRA 72 65 68 67 66 67 76 70
    MP3A3SR 73 65 67 67 65 66 77 71
    MP3C5SR 67 60 74 63 60 63 70 64
    MP3C1SR 67 59 73 63 60 62 70 65
    MP3G8SR 66 60 73 63 61 63 71 64
    MP3D3BR 65 58 73 64 60 63 68 67
    MP3H6SRA 71 69 71 71 68 70 82 70
    MP3B4SRA 71 69 71 71 68 70 82 70
    MP4E4BR 72 70 71 71 68 70 80 71
    MP4H8SR 70 67 69 70 67 70 79 71
    MP2F6SR 69 66 67 69 68 67 79 69
    MP3D1BR 68 66 67 71 69 69 79 70
    MP2B5BR 63 84 65 63 63 62 70 65
    MP2C1BR 61 85 65 64 63 62 70 65
    MP4A12SR 61 84 64 63 63 62 70 65
    MP3F4SRA 72 63 67 68 65 65 76 71
    MP3D3BR 70 64 66 66 64 64 75 69
    MP3E5BR 72 64 67 68 65 66 77 71
    MP3C7SRA 72 64 68 68 66 66 78 71
    MP2F1BR 70 64 68 65 64 64 74 67
    MP2C5BR 69 63 67 64 62 63 73 67
    MP2C10BR 67 66 69 68 64 68 74 73
    MP2G5SR 67 65 67 66 64 66 73 73
    MP3B1SRA 67 60 67 69 68 69 69 65
    MP2F10SR 67 65 71 66 65 67 77 68
    MP3A7SRA 68 63 71 65 65 67 77 69
    MP4C10SR 64 58 65 64 63 66 66 63
    MP4D5BR X 64 69 68 65 67 76 73
    MP3F1SRA — X 65 64 64 63 71 68
    MP6D6BR — — X 70 65 70 77 73
    MP6B1BR — — — X 78 81 76 71
    MP6A8BR — — — — X 75 74 66
    MP6B12BR — — — — — X 73 68
    MP6C11BR X 77
    MP6B10BR X
  • TABLE 15
    Fractional homologies between anti-vWF VHHs of the Invention.
    SEQ C37 C37-hum AM-2-75 22-2L-34 22-4L-16 T76 AM-4-15-3 A50 I53 Z29 M53 2A1-4L-79
    C37 1.00 0.95 0.99 0.59 0.68 0.63 0.63 0.65 0.59 0.57 0.59 0.57
    C37-hum — 1.00 0.94 0.59 0.68 0.63 0.63 0.65 0.58 0.57 0.60 0.59
    AM-2-75 — — 1.00 0.60 0.68 0.64 0.64 0.66 0.59 0.57 0.60 0.58
    22-2L-34 — — — 1.00 0.77 0.61 0.64 0.71 0.66 0.64 0.64 0.67
    22-4L-16 — — — — 1.00 0.71 0.70 0.80 0.70 0.73 0.69 0.70
    T76 — — — — — 1.00 0.77 0.68 0.59 0.62 0.61 0.61
    AM-4-15-3 — — — — — — 1.00 0.66 0.65 0.61 0.62 0.63
    A50 — — — — — — — 1.00 0.67 0.70 0.66 0.67
    I53 — — — — — — — — 1.00 0.63 0.69 0.70
    Z29 — — — — — — — — — 1.00 0.64 0.64
    M53 — — — — — — — — — — 1.00 0.70
    2A1-4L-79 — — — — — — — — — — — 1.00
    2A1-4L-129 — — — — — — — — — — — —
    2A1-4L-34 — — — — — — — — — — — —
    2A1-4L-78 — — — — — — — — — — — —
    2LA1-15 — — — — — — — — — — — —
    3P1-31 — — — — — — — — — — — —
    3L-41 — — — — — — — — — — — —
    3P2-31 — — — — — — — — — — — —
    C37-3 — — — — — — — — — — — —
    C37-4 — — — — — — — — — — — —
    C37-8 — — — — — — — — — — — —
    C37-10 — — — — — — — — — — — —
    SEQ 2A1-4L-129 2A1-4L-34 2A1-4L-78 2LA1-15 3P1-31 3L-41 3P2-31 C37-3 C37-4 C37-8 C37-10
    C37 0.61 0.59 0.62 0.61 0.66 0.63 0.60 0.97 0.96 0.93 0.91
    C37-hum 0.61 0.60 0.62 0.62 0.66 0.63 0.59 0.97 0.98 0.98 0.96
    AM-2-75 0.62 0.60 0.62 0.62 0.67 0.64 0.60 0.96 0.95 0.92 0.92
    22-2L-34 0.70 0.70 0.65 0.65 0.66 0.63 0.63 0.59 0.59 0.58 0.58
    22-4L-16 0.73 0.72 0.70 0.68 0.73 0.69 0.71 0.67 0.67 0.68 0.68
    T76 0.62 0.61 0.65 0.60 0.69 0.65 0.65 0.62 0.62 0.61 0.61
    AM-4-15-3 0.65 0.65 0.62 0.67 0.69 0.68 0.62 0.63 0.63 0.62 0.62
    A50 0.70 0.67 0.68 0.68 0.69 0.67 0.69 0.64 0.64 0.64 0.64
    I53 0.72 0.72 0.64 0.65 0.66 0.65 0.63 0.58 0.58 0.56 0.56
    Z29 0.67 0.68 0.71 0.64 0.63 0.61 0.66 0.56 0.56 0.56 0.56
    M53 0.70 0.72 0.67 0.60 0.64 0.64 0.69 0.59 0.59 0.58 0.60
    2A1-4L-79 0.88 0.85 0.66 0.63 0.64 0.62 0.62 0.57 0.57 0.57 0.57
    2A1-4L-129 1.00 0.88 0.70 0.65 0.67 0.64 0.64 0.61 0.61 0.60 0.60
    2A1-4L-34 — 1.00 0.66 0.64 0.65 0.64 0.62 0.58 0.58 0.58 0.58
    2A1-4L-78 — — 1.00 0.63 0.65 0.62 0.70 0.62 0.62 0.60 0.60
    2LA1-15 — — — 1.00 0.65 0.62 0.60 0.60 0.61 0.60 0.60
    3P1-31 — — — — 1.00 0.89 0.67 0.65 0.65 0.64 0.64
    3L-41 — — — — — 1.00 0.65 0.63 0.63 0.62 0.62
    3P2-31 — — — — — — 1.00 0.58 0.58 0.57 0.57
    C37-3 — — — — — — — 1.00 0.99 0.95 0.94
    C37-4 — — — — — — — — 1.00 0.96 0.95
    C37-8 — — — — — — — — — 1.00 0.98
    C37-10 — — — — — — — — — — 1.00

Claims (64)

1. A polypeptide construct comprising:
at least one single domain antibody directed against a therapeutic and/or diagnostic target, and
at least one single domain antibody directed against a serum protein.
2. A polypeptide construct according to claim 1 wherein:
the number of anti-target single domain antibodies is at least two, and
at least two anti-target single domain antibodies do not share the same sequence, or all the anti-target single domain antibodies share the same sequence.
3. A polypeptide construct according to claim 1 wherein:
the number of anti-serum protein single domain antibodies is at least two, and
at least two anti-serum-protein single domain antibodies do not share the same sequence, or all the anti-serum-protein single domain antibodies share the same sequence.
4. A polypeptide construct according to claim 1 wherein the at least one single domain antibody is a Camelidae VHHs antibody.
5. A polypeptide construct according to claim 1 wherein the at least one single domain antibody is a humanised Camelidae VHHs antibody.
6. A polypeptide construct according to claim 1 wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.
7. A polypeptide construct according to claim 1 wherein the at least one single domain anti-serum protein antibody corresponds to a sequence represented by any of SEQ ID NOS: 1 to 4, and 28 to 40.
8. A polypeptide construct according to claim 1 wherein the target is TNF-alpha.
9. A polypeptide construct according to claim 7 corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.
10. (canceled)
11. A nucleic acid encoding a polypeptide construct according to claim 9.
12. (canceled)
13. A method for treatment, prevention and/or alleviation of disorders relating to inflammatory processes comprising administering to a subject in need of such treatment a polypeptide construct according to claim 8.
14. (canceled)
15. The method according to claim 13 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
16. A polypeptide construct according to claim 1 wherein a the target is vWF
17. A polypeptide construct according to claim 1 wherein the target is collagen.
18. A polypeptide construct according to claim 16 wherein at least one anti-target single domain antibody is anti-vWF VHHs.
19. A polypeptide construct according to claim 18 corresponding to the sequence represented by any of SEQ ID NOS: 19 to 21.
20. (canceled)
21. A nucleic acid encoding a polypeptide construct according to claim 19.
22. A method for treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof comprising administering to a subject in need of such treatment A polypeptide construct according to claim 16.
23. (canceled)
24. A method according to claim 22 wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.
25. A method according to claim 22 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
26. A polypeptide construct according to claim 1 wherein the target is IgE.
27. A polypeptide construct according to claim 26 wherein the at least one anti-target single domain antibody is anti-IgE VHHs.
28. A polypeptide construct according to claim 26 corresponding to the sequence represented by any of SEQ ID NOS: 22 to 24.
29. (canceled)
30. A nucleic acid encoding a polypeptide construct according to claim 28.
31. (canceled)
32. A method for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions comprising administering to a subject in need of such treatment a polypeptide construct according to claim 26.
33. A method according to claim 32 wherein said disorders or conditions are any of hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.
34. A method according to claim 32 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
35. A polypeptide construct according to claim 1 wherein the target is IFN-gamma.
36. A polypeptide construct according to claim 35 wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.
37. A polypeptide construct according to claim 35 corresponding to a sequence represented by SEQ ID NOs: 25 to 27.
38. (canceled)
39. A nucleic acid encoding a polypeptide construct according to claim 37.
40. (canceled)
41. A method for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active comprising administering to a subject in need of such treatment a polypeptide construct according to claim 35.
42. A method according to claim 41 wherein said disorders or conditions are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
43. A method according to claim 41 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
44. A composition comprising a polypeptide construct according to claim 8 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
45. A composition comprising a polypeptide construct according to claim 16 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
46. A composition comprising a polypeptide construct according to claim 26 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.
47. A polypeptide construct according to claim 1 directed against a single target wherein said target is involved in a disease process.
48. (canceled)
49. A nucleic acid encoding a polypeptide construct according to claim 47.
50.-52. (canceled)
53. A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation comprising administering to a subject in need of such treatment the polypeptide construct according to claim 47.
54. (canceled)
55. A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time comprising administering to a subject in need of such treatment the polypeptide construct according to claim 47.
56. A according to any of claims 53 and 55, wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.
57. (canceled)
58. A method of producing a polypeptide according to claim 1 comprising
(a) culturing host cells comprising nucleic acid capable of encoding a polypeptide according to claim 1, under conditions allowing the expression of the polypeptide, and,
(b) recovering the produced polypeptide from the culture.
59. A method according to claim 58, wherein said host cells are bacterial or yeast.
60. A method for prolonging the half-life of a single domain antibody in the blood stream of a subject, said antibody directed against a therapeutic and/or diagnostic target, comprising joining thereto one or more single domain antibodies directed against a serum protein.
61. A method according to claim 60 wherein said anti-target single domain antibodies do not share the same sequence.
62. A method according to claim 60 wherein said anti-serum protein single domain antibodies do not share the same sequence.
63. A method according to claim 60 wherein said single domain antibodies are Camelidae VHH antibodies.
64. A method according to claim 60 wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.
65. A method according to claim 60 wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOs: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.
66. A composition comprising a polypeptide according to claim 1 or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.
US10/534,349 2002-11-08 2003-11-07 Stabilized single domain antibodies Abandoned US20070178082A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/534,349 US20070178082A1 (en) 2002-11-08 2003-11-07 Stabilized single domain antibodies

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US42506302P 2002-11-08 2002-11-08
US42507302P 2002-11-08 2002-11-08
EP03447005 2003-01-10
EP03447005.4 2003-01-10
EPPCT/EP03/06581 2003-06-23
EPPCT/EP03/06581 2003-06-23
EPPCT/EP03/07313 2003-07-08
EPPCT/EP2003/007313 2003-07-08
US10/534,349 US20070178082A1 (en) 2002-11-08 2003-11-07 Stabilized single domain antibodies
PCT/BE2003/000193 WO2004041865A2 (en) 2002-11-08 2003-11-07 Stabilized single domain antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/BE2003/000193 A-371-Of-International WO2004041865A2 (en) 2002-11-08 2003-11-07 Stabilized single domain antibodies

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/804,543 Continuation US20090238829A1 (en) 2002-11-08 2007-05-18 Stabilized single domain antibodies

Publications (1)

Publication Number Publication Date
US20070178082A1 true US20070178082A1 (en) 2007-08-02

Family

ID=32893039

Family Applications (11)

Application Number Title Priority Date Filing Date
US10/534,349 Abandoned US20070178082A1 (en) 2002-11-08 2003-11-07 Stabilized single domain antibodies
US10/534,292 Abandoned US20060115470A1 (en) 2002-11-08 2003-11-07 Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
US10/541,708 Active 2027-05-22 US9028816B2 (en) 2003-01-10 2004-01-09 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von Willebrand factor
US12/487,684 Expired - Lifetime US9243065B2 (en) 2002-11-08 2009-06-19 Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US12/488,077 Abandoned US20100040613A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for sublingual administration
US12/487,895 Abandoned US20100003249A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for topical administration
US12/487,949 Abandoned US20090324512A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for nasal administration
US12/487,712 Abandoned US20100003248A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for rectal and/or vaginal administration
US13/078,184 Abandoned US20110184145A1 (en) 2002-11-08 2011-04-01 Method of administering therapeutic polypeptides, and polypeptides therefor
US14/669,025 Expired - Lifetime US10112989B2 (en) 2003-01-10 2015-03-26 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von Willebrand factor
US16/142,063 Expired - Fee Related US11034755B2 (en) 2003-01-10 2018-09-26 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von willebrand factor

Family Applications After (10)

Application Number Title Priority Date Filing Date
US10/534,292 Abandoned US20060115470A1 (en) 2002-11-08 2003-11-07 Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
US10/541,708 Active 2027-05-22 US9028816B2 (en) 2003-01-10 2004-01-09 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von Willebrand factor
US12/487,684 Expired - Lifetime US9243065B2 (en) 2002-11-08 2009-06-19 Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US12/488,077 Abandoned US20100040613A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for sublingual administration
US12/487,895 Abandoned US20100003249A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for topical administration
US12/487,949 Abandoned US20090324512A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for nasal administration
US12/487,712 Abandoned US20100003248A1 (en) 2002-11-08 2009-06-19 Polypeptide constructs for rectal and/or vaginal administration
US13/078,184 Abandoned US20110184145A1 (en) 2002-11-08 2011-04-01 Method of administering therapeutic polypeptides, and polypeptides therefor
US14/669,025 Expired - Lifetime US10112989B2 (en) 2003-01-10 2015-03-26 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von Willebrand factor
US16/142,063 Expired - Fee Related US11034755B2 (en) 2003-01-10 2018-09-26 Polypeptides and polypeptide constructs comprising single domain antibodies directed against von willebrand factor

Country Status (19)

Country Link
US (11) US20070178082A1 (en)
EP (10) EP2284192A3 (en)
JP (5) JP2006524036A (en)
KR (2) KR20050092029A (en)
CN (3) CN102584997A (en)
AT (1) ATE517922T1 (en)
AU (2) AU2004204262B2 (en)
BR (1) BRPI0406694B8 (en)
CA (4) CA2505326A1 (en)
DK (1) DK2316852T3 (en)
ES (4) ES2655912T3 (en)
IL (1) IL168357A (en)
MX (2) MXPA05004955A (en)
NO (1) NO337265B1 (en)
NZ (5) NZ540196A (en)
PT (1) PT2316852E (en)
RU (2) RU2357974C2 (en)
WO (4) WO2004041863A2 (en)
ZA (1) ZA200504996B (en)

Cited By (114)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050054001A1 (en) * 2001-10-24 2005-03-10 Serge Muyldermans Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
US20050260155A1 (en) * 2004-05-18 2005-11-24 Gopala Kovvali Compositions and methods for treatment of ulcerative colitis
US20060034845A1 (en) * 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
US20070269422A1 (en) * 2006-05-17 2007-11-22 Ablynx N.V. Serum albumin binding proteins with long half-lives
US20080260757A1 (en) * 2004-06-01 2008-10-23 Domantis Limited Bispecific Fusion Antibodies With Enhanced Serum Half-Life
US20080269070A1 (en) * 2007-04-27 2008-10-30 Dow Global Technologies, Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
US20090238829A1 (en) * 2002-11-08 2009-09-24 Ablynx N.V. Stabilized single domain antibodies
US20090324512A1 (en) * 2002-11-08 2009-12-31 Ablynx N.V. Polypeptide constructs for nasal administration
US20100003253A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
WO2010060095A1 (en) 2008-11-24 2010-05-27 Bristol-Myers Squibb Company Bispecific egfr/igfir binding molecules
US20100137162A1 (en) * 2007-04-27 2010-06-03 Pfenex Biopharmaceuticals, Inc. Method for Rapidly Screening Microbial Hosts to Identify Certain Strains with Improved Yield and/or Quality in the Expression of Heterologous Proteins
US20100172894A1 (en) * 2008-10-29 2010-07-08 Wyeth Methods for purification of single domain antigen binding molecules
US20100297111A1 (en) * 2005-05-21 2010-11-25 Els Anna Alice Beirnaert Nanobodies against tumor necrosis factor-alpha
US20100330080A1 (en) * 2008-07-02 2010-12-30 Torsten Dreier Antigen binding polypeptides
WO2011051327A3 (en) * 2009-10-30 2011-06-30 Novartis Ag Small antibody-like single chain proteins
WO2011103105A1 (en) 2010-02-18 2011-08-25 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind il-23
WO2011130354A1 (en) 2010-04-13 2011-10-20 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind pcsk9
WO2011150133A2 (en) 2010-05-26 2011-12-01 Bristol-Myers Squibb Company Fibronectin based scaffold proteins having improved stability
US8188223B2 (en) 2005-05-18 2012-05-29 Ablynx N.V. Serum albumin binding proteins
WO2012088006A1 (en) 2010-12-22 2012-06-28 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind il-23
WO2013067029A2 (en) 2011-10-31 2013-05-10 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
WO2013119903A1 (en) 2012-02-10 2013-08-15 Research Corporation Technologies, Inc. Fusion proteins comprising immunoglobulin constant domain-derived scaffolds
WO2013138338A2 (en) 2012-03-12 2013-09-19 Massachusetts Institute Of Technology Methods for treating tissue damage associated with ischemia with apoliporotein d
WO2013177187A2 (en) 2012-05-22 2013-11-28 Massachusetts Institute Of Technology Synergistic tumor treatment with extended-pk il-2 and therapeutic agents
US8603824B2 (en) 2004-07-26 2013-12-10 Pfenex, Inc. Process for improved protein expression by strain engineering
WO2014120891A2 (en) 2013-02-01 2014-08-07 Bristol-Myers Squibb Company Fibronectin based scaffold proteins
WO2014165093A2 (en) 2013-03-13 2014-10-09 Bristol-Myers Squibb Company Fibronectin based scaffold domains linked to serum albumin or a moiety binding thereto
EP2799448A1 (en) 2008-05-22 2014-11-05 Bristol-Myers Squibb Company Multivalent fibronectin based scaffold domain proteins
WO2016025642A1 (en) 2014-08-12 2016-02-18 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2 and integrin-binding-fc-fusion protein
WO2016025647A1 (en) 2014-08-12 2016-02-18 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2, a therapeutic antibody, and a cancer vaccine
US9284361B2 (en) 2010-11-26 2016-03-15 Molecular Partners Ag Designed repeat proteins binding to serum albumin
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
WO2016086021A1 (en) 2014-11-25 2016-06-02 Bristol-Myers Squibb Company Novel pd-l1 binding polypeptides for imaging
US9393304B2 (en) 2008-10-29 2016-07-19 Ablynx N.V. Formulations of single domain antigen binding molecules
WO2016171980A1 (en) 2015-04-24 2016-10-27 Bristol-Myers Squibb Company Polypeptides targeting hiv fusion
US9708412B2 (en) 2015-05-21 2017-07-18 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US9920115B2 (en) 2016-05-20 2018-03-20 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US10041102B2 (en) 2002-10-08 2018-08-07 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
US10066016B2 (en) 2016-05-20 2018-09-04 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
WO2019036605A2 (en) 2017-08-17 2019-02-21 Massachusetts Institute Of Technology Multiple specificity binders of cxc chemokines and uses thereof
US10350266B2 (en) 2017-01-10 2019-07-16 Nodus Therapeutics, Inc. Method of treating cancer with a multiple integrin binding Fc fusion protein
WO2019154985A1 (en) 2018-02-12 2019-08-15 Biontech Rna Pharmaceuticals Gmbh Treatment using cytokine encoding rna
US10543271B2 (en) 2017-05-12 2020-01-28 Harpoon Therapeutics, Inc. Mesothelin binding proteins
WO2020020783A1 (en) 2018-07-24 2020-01-30 Biontech Rna Pharmaceuticals Gmbh Il2 agonists
US10603358B2 (en) 2017-01-10 2020-03-31 Nodus Therapeutics Combination tumor treatment with an integrin-binding-Fc fusion protein and immune stimulator
WO2020068261A1 (en) 2018-09-28 2020-04-02 Massachusetts Institute Of Technology Collagen-localized immunomodulatory molecules and methods thereof
US10633438B2 (en) 2015-03-31 2020-04-28 Vhsquared Limited Polypeptides
WO2020154032A1 (en) 2019-01-23 2020-07-30 Massachusetts Institute Of Technology Combination immunotherapy dosing regimen for immune checkpoint blockade
US10730954B2 (en) 2017-05-12 2020-08-04 Harpoon Therapeutics, Inc. MSLN targeting trispecific proteins and methods of use
WO2020161224A1 (en) 2019-02-08 2020-08-13 Biontech Cell & Gene Therapies Gmbh Treatment involving car-engineered t cells and cytokines
WO2020187848A1 (en) 2019-03-18 2020-09-24 Biontech Cell & Gene Therapies Gmbh Lnterleukin-2 receptor (il2r) and interleukin-2 (il2) variants for specific activation of immune effector cells
WO2020201448A1 (en) 2019-04-05 2020-10-08 Biontech Rna Pharmaceuticals Gmbh Treatment involving interleukin-2 (il2) and interferon (ifn)
US10815311B2 (en) 2018-09-25 2020-10-27 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use
EP3733698A1 (en) 2015-09-23 2020-11-04 Bristol-Myers Squibb Company Glypican-3 binding fibronectin based scafflold molecules
US10844134B2 (en) 2016-11-23 2020-11-24 Harpoon Therapeutics, Inc. PSMA targeting trispecific proteins and methods of use
US10849973B2 (en) 2016-11-23 2020-12-01 Harpoon Therapeutics, Inc. Prostate specific membrane antigen binding protein
WO2020260270A1 (en) 2019-06-24 2020-12-30 Biontech Rna Pharmaceuticals Gmbh Il2 agonists
US10927180B2 (en) 2017-10-13 2021-02-23 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
WO2021058091A1 (en) 2019-09-24 2021-04-01 Biontech Rna Pharmaceuticals Gmbh Treatment involving therapeutic antibody and interleukin-2 (il2)
EP3835310A1 (en) 2012-09-13 2021-06-16 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind to myostatin
WO2021127495A1 (en) 2019-12-20 2021-06-24 Regeneron Pharmaceuticals, Inc. Novel il2 agonists and methods of use thereof
WO2021130223A1 (en) 2019-12-23 2021-07-01 Biontech Cell & Gene Therapies Gmbh Treatment involving immune effector cells genetically modified to express antigen receptors
WO2021130225A1 (en) 2019-12-27 2021-07-01 Biontech Cell & Gene Therapies Gmbh In vitro and in vivo gene delivery to immune effector cells using nanoparticles functionalized with designed ankyrin repeat proteins (darpins)
WO2021185775A1 (en) 2020-03-16 2021-09-23 Biontech Cell & Gene Therapies Gmbh Antigen-specific t cell receptors and t cell epitopes
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
WO2021198258A1 (en) 2020-03-31 2021-10-07 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination
WO2021231447A1 (en) 2020-05-12 2021-11-18 Regeneron Pharmaceuticals, Inc. Novel il10 agonists and methods of use thereof
US11180563B2 (en) 2020-02-21 2021-11-23 Harpoon Therapeutics, Inc. FLT3 binding proteins and methods of use
WO2022135666A1 (en) 2020-12-21 2022-06-30 BioNTech SE Treatment schedule for cytokine proteins
WO2022136257A1 (en) 2020-12-21 2022-06-30 BioNTech SE Therapeutic rna for treating cancer
WO2022136266A1 (en) 2020-12-21 2022-06-30 BioNTech SE Therapeutic rna for treating cancer
WO2022218891A2 (en) 2021-04-12 2022-10-20 BioNTech SE Rna compositions comprising a buffer substance and methods for preparing, storing and using the same
WO2022223617A1 (en) 2021-04-20 2022-10-27 BioNTech SE Virus vaccine
US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
WO2023004282A2 (en) 2021-07-19 2023-01-26 Regeneron Pharmaceuticals, Inc. Il12 receptor agonists and methods of use thereof
WO2023022965A2 (en) 2021-08-16 2023-02-23 Regeneron Pharmaceuticals, Inc. Novel il27 receptor agonists and methods of use thereof
WO2023052531A1 (en) 2021-09-30 2023-04-06 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination and pd-1 axis binding antagonists
US11623952B2 (en) 2019-06-21 2023-04-11 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
WO2023067193A2 (en) 2021-10-22 2023-04-27 BioNTech SE Compositions for administration of different doses of rna
WO2023066496A1 (en) 2021-10-21 2023-04-27 BioNTech SE Coronavirus vaccine
WO2023083434A1 (en) 2021-11-09 2023-05-19 BioNTech SE Rna encoding peptidoglycan hydrolase and use thereof for treating bacterial infection
WO2023086812A1 (en) 2021-11-11 2023-05-19 Regeneron Pharmaceuticals, Inc. Cd20-pd1 binding molecules and methods of use thereof
US11667719B2 (en) 2019-06-21 2023-06-06 Sorriso Pharmaceuticals, Inc. VHH immunoglobulin chain variable domain that binds to IL-7R and methods of use thereof for treating autoimmune and/or inflammatory diseases
US11684677B2 (en) 2016-09-30 2023-06-27 Sorriso Pharmaceuticals, Inc. Compositions
WO2023126053A1 (en) 2021-12-28 2023-07-06 BioNTech SE Lipid-based formulations for administration of rna
WO2023166099A1 (en) 2022-03-01 2023-09-07 BioNTech SE RNA LIPID NANOPARTICLES (LNPs) COMPRISING A POLYOXAZOLINE AND/OR POLYOXAZINE POLYMER
WO2023194508A1 (en) 2022-04-05 2023-10-12 BioNTech SE Nucleic acid compositions comprising a multivalent anion, such as an inorganic polyphosphate, and methods for preparing, storing and using the same
WO2023215810A1 (en) * 2022-05-05 2023-11-09 Inhibrx, Inc. Albumin-binding polypeptides and uses thereof
WO2024002985A1 (en) 2022-06-26 2024-01-04 BioNTech SE Coronavirus vaccine
WO2024017479A1 (en) 2022-07-21 2024-01-25 BioNTech SE Multifunctional cells transiently expressing an immune receptor and one or more cytokines, their use and methods for their production
WO2024028445A1 (en) 2022-08-03 2024-02-08 BioNTech SE Rna for preventing or treating tuberculosis
WO2024027910A1 (en) 2022-08-03 2024-02-08 BioNTech SE Rna for preventing or treating tuberculosis
WO2024028325A1 (en) 2022-08-01 2024-02-08 BioNTech SE Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions
WO2024151978A1 (en) 2023-01-13 2024-07-18 Regeneron Pharmaceuticals, Inc. Il12 receptor agonists and methods of use thereof
WO2024153324A1 (en) 2023-01-18 2024-07-25 BioNTech SE Rna formulations for pharmaceutical use
WO2024216212A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024216214A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024213776A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024233926A1 (en) 2023-05-10 2024-11-14 Regeneron Pharmaceuticals, Inc. Cd20-pd1 binding molecules and methods of use thereof
US12173054B2 (en) 2015-03-31 2024-12-24 Sorriso Pharmaceuticals, Inc. Polypeptides
WO2025006676A2 (en) 2023-06-27 2025-01-02 Firecyte Therapeutics, Inc. Insulin-like growth factor binding protein like 1 (igfbpl1) compositions and methods of use thereof
US12195544B2 (en) 2018-09-21 2025-01-14 Harpoon Therapeutics, Inc. EGFR binding proteins and methods of use
WO2025021668A1 (en) 2023-07-21 2025-01-30 BioNTech SE Agents and methods for targeted delivery of cytokines to immune cells
WO2025026866A1 (en) 2023-07-28 2025-02-06 BioNTech SE Rna therapeutics with reduced toxicity
US12234279B2 (en) 2015-03-31 2025-02-25 Sorriso Pharmaceuticals, Inc. Peptide construct having a protease-cleavable linker
US12234297B2 (en) 2023-07-13 2025-02-25 Phylaxis Bioscience, Llc IgE binding proteins and uses thereof
WO2025101851A1 (en) 2023-11-10 2025-05-15 Regeneron Pharmaceuticals, Inc. Engineered alpha klotho polypeptides and uses thereof
WO2025106754A1 (en) 2023-11-15 2025-05-22 BioNTech SE Coronavirus vaccine
US12415860B2 (en) 2018-05-14 2025-09-16 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
WO2025255508A1 (en) 2024-06-07 2025-12-11 Regeneron Pharmaceuticals, Inc. Engineered alpha klotho polypeptides and uses thereof
US12516128B2 (en) 2019-05-14 2026-01-06 Harpoon Therapeutics, Inc. EpCAM binding proteins and methods of use
US12570736B2 (en) 2019-06-21 2026-03-10 Sorriso Pharmaceuticals, Inc. Compositions
WO2026082899A2 (en) 2024-10-16 2026-04-23 BioNTech SE Rna for preventing or treating tuberculosis

Families Citing this family (472)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090311267A1 (en) * 1999-08-10 2009-12-17 University Of Wurzburg Inhibition of VWF - GPIb/V/IX interaction and platelet-collagen interaction for prevention and treatment of cerebral attacks
US20060073141A1 (en) * 2001-06-28 2006-04-06 Domantis Limited Compositions and methods for treating inflammatory disorders
US9028822B2 (en) 2002-06-28 2015-05-12 Domantis Limited Antagonists against TNFR1 and methods of use therefor
EP1600459A3 (en) 2002-06-28 2005-12-07 Domantis Limited Ligand
US9321832B2 (en) 2002-06-28 2016-04-26 Domantis Limited Ligand
AU2003251238A1 (en) 2002-08-07 2004-02-25 Umc Utrecht Holding B.V. Modulation of platelet adhesion based on the surface exposed beta-switch loop of platelet glycoprotein ib-alpha
US20060034833A1 (en) * 2002-11-08 2006-02-16 Els Beirnaert Single domain antibodies directed against interferron-gamma and uses therefor
SI1639011T1 (en) 2003-06-30 2009-04-30 Domantis Ltd Pegylated single domain antibodies (dAb)
PT1687338E (en) * 2003-11-07 2011-01-20 Ablynx Nv Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses thereof
WO2005077980A2 (en) * 2004-02-13 2005-08-25 Bioceros Bv Soluble tcr-like molecules and their uses
JP2008504356A (en) * 2004-06-30 2008-02-14 ドマンティス リミテッド Compositions and methods for treating inflammatory diseases
US7563443B2 (en) 2004-09-17 2009-07-21 Domantis Limited Monovalent anti-CD40L antibody polypeptides and compositions thereof
EP1841452B8 (en) 2004-10-08 2014-06-11 Domantis Limited Single domain antibodies against tnfr1 and methods of use therefor
WO2006040153A2 (en) * 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
BRPI0518761A2 (en) * 2004-12-02 2008-12-09 Domantis Ltd drug fusion, drug conjugate, Recombinant Nucleic Acid, Nucleic Acid Construction, Host Cell, Method for Producing a Drug Fusion, Pharmaceutical Composition, Drug, Method of Treatment and / or Prevention of a Condition in a Patient, Delay Method or prevention of disease progression, and a method for decreasing food absorption by a patient
CA2588892A1 (en) * 2004-12-02 2006-06-08 Dormantis Limited Anti-il-1r1 single domain antibodies and therapeutic uses
US20090111745A1 (en) * 2004-12-02 2009-04-30 Domantis Limited Plad Domain Peptides With Increased Serum Half Life Due To Conjugation To Domain Antibodies
CA2589839C (en) * 2004-12-02 2016-04-05 Unilever N.V. Method for affinity purification
US20090298195A1 (en) 2005-01-05 2009-12-03 F-Star Biotechnologische Forschungs-Und Entwicklun Gseges M.B.H. Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions
CA2593328C (en) 2005-01-14 2018-02-06 Ablynx N.V. Methods and assays for distinguishing between different forms of diseases and disorders characterized by thrombocytopenia and/or by spontaneous interaction between von willebrand factor (vwf) and platelets
JP4913034B2 (en) * 2005-02-14 2012-04-11 アルフレッサファーマ株式会社 ADAMTS13 activity assay antibody and activity assay method
WO2006097689A2 (en) * 2005-03-18 2006-09-21 Domantis Limited Antibodies against candida antigens
AU2012200682B2 (en) * 2005-05-18 2014-11-27 Ablynx Nv Improved NanobodiesTM against Tumor Necrosis Factor-alpha
MX2007014564A (en) * 2005-05-20 2008-02-07 Ablynx Nv VHH ANTIBODIES OF SINGLE DOMAIN AGAINST VON WILLEBRAND FACTOR.
US9017662B2 (en) 2005-08-30 2015-04-28 Actogenix N.V. Anti-TNF alpha producing lactic acid bacteria for the treatment of chronic enterocolitis
WO2007026972A2 (en) * 2005-09-01 2007-03-08 Canon Kabushiki Kaisha Binding protein molecule
US20100323905A1 (en) * 2005-09-23 2010-12-23 Academisch Ziekenhuis Leiden Vhh for the Diagnosis, Prevention and Treatment of Diseases Associated with Protein Aggregates
EP1940880A2 (en) * 2005-10-14 2008-07-09 Novo Nordisk A/S Treating diabetes using inhibitors of il-1
GB0521958D0 (en) * 2005-10-27 2005-12-07 Ares Trading Sa vWFA, collagen and kunitz domain containing protein
AR056806A1 (en) 2005-11-14 2007-10-24 Amgen Inc RANKL- PTH / PTHRP ANTIBODY CHEMICAL MOLECULES
NZ568992A (en) * 2005-11-17 2012-01-12 Biogen Idec Inc Platelet aggregation assays comprising platelet activiating agents and agents that cross-links the CD40L-binding moiety
JP2007172129A (en) * 2005-12-20 2007-07-05 Sony Corp Nonvolatile memory access control device and nonvolatile memory control system
EP2004690A2 (en) * 2006-03-13 2008-12-24 Ablynx N.V. Amino acid sequences directed against il-6 and polypeptides comprising the same for the treatment of diseases and disorders associated with il-6-mediated signalling
EP1999147A1 (en) * 2006-03-27 2008-12-10 Ablynx N.V. Medical delivery device for therapeutic proteins based on single domain antibodies
WO2007112940A2 (en) * 2006-03-31 2007-10-11 Ablynx N.V. Albumin-derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic compounds and entities, and constructs comprising the same
EP2010568A1 (en) * 2006-04-14 2009-01-07 Ablynx N.V. Dp-78-like nanobodies
AT503889B1 (en) 2006-07-05 2011-12-15 Star Biotechnologische Forschungs Und Entwicklungsges M B H F MULTIVALENT IMMUNE LOBULINE
AT503902B1 (en) 2006-07-05 2008-06-15 F Star Biotech Forsch & Entw METHOD FOR MANIPULATING IMMUNE LOBULINS
US20080015145A1 (en) 2006-07-11 2008-01-17 Maria Gyongyossy-Issa Mimotope receptors and inhibitors for platelet-platelet and platelet-endothelium interactions
CN101646689A (en) * 2006-09-08 2010-02-10 埃博灵克斯股份有限公司 Serum albumin binding proteins with long half-lives
SI3028716T1 (en) 2006-10-10 2021-01-29 Regenesance B.V. Complement inhibition for improved nerve regeneration
JP2010505435A (en) * 2006-10-11 2010-02-25 アブリンクス エン．ヴェー． Amino acid sequences that bind to serum proteins essentially independently of pH, compounds containing them, and uses thereof
WO2008049881A2 (en) * 2006-10-25 2008-05-02 Umc Utrecht Holding Bv Polypeptides and pharmaceutical compositions comprising the same for the prevention and treatment of complications associated with infectious diseases
US20100129354A1 (en) * 2006-10-27 2010-05-27 Ablynx N.V. Intranasal delivery of polypeptides and proteins
GB0621513D0 (en) 2006-10-30 2006-12-06 Domantis Ltd Novel polypeptides and uses thereof
WO2008068280A1 (en) * 2006-12-05 2008-06-12 Ablynx N.V. Peptides capable of binding to serum proteins
WO2008071751A1 (en) * 2006-12-14 2008-06-19 Actogenix N.V. Delivery of binding molecules to induce immunomodulation
CA2673331A1 (en) 2006-12-19 2008-06-26 Ablynx N.V. Amino acid sequences directed against gpcrs and polypeptides comprising the same for the treatment of gpcr-related diseases and disorders
AU2007336243B2 (en) * 2006-12-19 2012-07-26 Ablynx N.V. Amino acid sequences directed against a metalloproteinase from the ADAM family and polypeptides comprising the same for the treatment of ADAM-related diseases and disorders
US9023352B2 (en) 2007-02-20 2015-05-05 Tufts University Methods, compositions and kits for treating a subject using a recombinant heteromultimeric neutralizing binding protein
SI2308514T1 (en) 2007-03-23 2013-09-30 To-Bbb Holding B.V. Conjugates for targeted drug delivery across the blood-brain barrier
WO2008121814A1 (en) * 2007-03-28 2008-10-09 Ats Medical, Inc. Method for inhibiting platelet interaction with biomaterial surfaces
WO2008124858A2 (en) * 2007-04-11 2008-10-23 F-Star Biotechnologische Forschungs- Und Entwicklungsges. M.B.H. Targeted receptor
KR101264473B1 (en) 2007-05-24 2013-05-29 아블린쓰 엔.브이. Amino acid sequences directed against rank-l and polypeptides comprising the same for the treatment of bone diseases and disorders
WO2008150431A1 (en) * 2007-05-29 2008-12-11 Yale University Il- 18 and protein kinase r inhibition for the treatment of copd
AU2008259590A1 (en) 2007-06-06 2008-12-11 Domantis Limited Methods for selecting protease resistant polypeptides
PL3241842T3 (en) 2007-06-26 2024-06-10 F-Star Therapeutics Limited Display of binding agents
AU2008270274B2 (en) 2007-07-03 2012-06-28 Ablynx N.V. Providing improved immunoglobulin sequences by mutating CDR and/or FR positions
US8629246B2 (en) * 2007-09-26 2014-01-14 Ucb Pharma S.A. Dual specificity antibody fusions
GB2453589A (en) 2007-10-12 2009-04-15 King S College London Protease inhibition
US20110027269A1 (en) 2007-11-27 2011-02-03 The Unversity of British Office 14-3-3 Antagonists for the Prevention and Treatment of Arthritis
WO2009068631A1 (en) 2007-11-27 2009-06-04 Ablynx N.V. Method for obtaining polypeptide constructs comprising two or more single domain antibodies
TW200938222A (en) * 2007-12-13 2009-09-16 Glaxo Group Ltd Compositions for pulmonary delivery
WO2009086400A2 (en) * 2007-12-28 2009-07-09 Baxter International Inc. Recombinant vwf formulations
EP2080770A1 (en) * 2008-01-21 2009-07-22 MorphoSys AG Proteinaceous binding molecules comprising purification tags
WO2009111507A1 (en) * 2008-03-03 2009-09-11 Dyax Corp. Metalloproteinase 12 binding proteins
CA2717015A1 (en) 2008-03-05 2009-09-11 Ablynx Nv Novel antigen binding dimer-complexes, methods of making and uses thereof
CA2718081A1 (en) * 2008-03-21 2009-09-24 Ablynx Nv Von willebrand factor specific binders and methods of use therefor
GB0805608D0 (en) * 2008-03-28 2008-04-30 Sec Dep For Environment Food & Detection method
GB0809069D0 (en) 2008-05-19 2008-06-25 Univ Leuven Kath Gene signatures
AU2009235467A1 (en) 2008-04-07 2009-10-15 Ablynx Nv Single variable domains against the Notch pathways
CN102089325A (en) 2008-04-17 2011-06-08 埃博灵克斯股份有限公司 Peptides capable of binding to serum proteins and compounds, constructs and polypeptides comprising the same
US9296810B2 (en) 2008-05-02 2016-03-29 Novartis Ag Fibronectin-based binding molecules and uses thereof
EP2113255A1 (en) 2008-05-02 2009-11-04 f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. Cytotoxic immunoglobulin
EP2700651B1 (en) 2008-07-18 2019-04-10 Bristol-Myers Squibb Company Compositions monovalent for CD28 binding and methods of use
WO2010035012A1 (en) 2008-09-26 2010-04-01 Ucb Pharma S.A. Biological products
CA2740561C (en) 2008-10-14 2021-01-19 National Research Council Of Canada Bsa-specific antibodies
WO2010043057A1 (en) * 2008-10-14 2010-04-22 National Research Counsil Of Canada Bsa-specific antibodies
JP2012506237A (en) * 2008-10-21 2012-03-15 ドマンティス リミテッド Ligand with binding specificity for DC-SIGN
AU2013202856B2 (en) * 2008-10-29 2016-02-11 Ablynx N.V. Formulations of single domain antigen binding molecules
WO2010063818A2 (en) 2008-12-05 2010-06-10 Glaxo Group Limited Methods for selecting protease resistant polypeptides
AU2010205627B2 (en) * 2009-01-14 2015-04-02 Ablynx Nv Pulmonary administration of immunoglobulin single variable domains and constructs thereof
EA022925B1 (en) 2009-02-19 2016-03-31 Глаксо Груп Лимитед Improved anti-tnfr1 polypeptides, antibody variable domains & antagonists
US9265834B2 (en) 2009-03-05 2016-02-23 Ablynx N.V. Stable formulations of polypeptides and uses thereof
WO2010100135A1 (en) 2009-03-05 2010-09-10 Ablynx N.V. Novel antigen binding dimer-complexes, methods of making/avoiding and uses thereof
US8748581B2 (en) 2009-04-10 2014-06-10 Ablynx N.V. Anti-IL-6R polypeptides and pharmaceutical compositions thereof
AU2010236256C1 (en) * 2009-04-16 2015-10-22 Abbvie Biotherapeutics Inc. Anti-TNF-alpha antibodies and their uses
HUE053480T2 (en) 2009-04-30 2021-06-28 Ablynx Nv Method for the production of domain antibodies
KR101852204B1 (en) 2009-06-05 2018-04-26 아블린쓰 엔.브이. Monovalent, bivalent and trivalent anti human respiratory syncytial virus (hrsv) nanobody constructs for the prevention and/or treatment of respiratory tract infections
US9150640B2 (en) 2009-07-10 2015-10-06 Ablynx N.V. Method for the production of variable domains
WO2011006914A2 (en) 2009-07-16 2011-01-20 Glaxo Group Limited Antagonists, uses & methods for partially inhibiting tnfr1
JP5426026B2 (en) 2009-07-28 2014-02-26 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Non-invasive in vivo optical imaging method
KR20120063475A (en) 2009-07-29 2012-06-15 글락소 그룹 리미티드 Anti-tgf-beta receptor type ii single domain antibodies
LT2805731T (en) 2009-09-03 2019-02-11 Ablynx N.V. Stable formulations of polypeptides and uses thereof
GB201005063D0 (en) 2010-03-25 2010-05-12 Ucb Pharma Sa Biological products
US20110195494A1 (en) 2009-10-02 2011-08-11 Boehringer Ingelheim International Gmbh Dll4-binging molecules
UY32920A (en) 2009-10-02 2011-04-29 Boehringer Ingelheim Int BISPECIFIC UNION MOLECULES FOR ANTI-ANGIOGENESIS THERAPY
WO2011042398A1 (en) 2009-10-09 2011-04-14 Ablynx Nv Immunoglobulin single variable domain directed against human cxcr4 and other cell associated proteins and methods to generate them
US9340605B2 (en) 2009-10-22 2016-05-17 Universiteit Twente VHH for application in tissue repair, organ regeneration, organ replacement and tissue engineering
TW201125877A (en) 2009-10-27 2011-08-01 Glaxo Group Ltd Stable anti-TNFR1 polypeptides, antibody variable domains & and antagonists
ES2860453T3 (en) 2009-10-30 2021-10-05 Novartis Ag Universal libraries of type III fibronectin underside binding domain
WO2011064382A1 (en) 2009-11-30 2011-06-03 Ablynx N.V. Improved amino acid sequences directed against human respiratory syncytial virus (hrsv) and polypeptides comprising the same for the prevention and/or treatment of respiratory tract infections
US20120321640A1 (en) 2009-12-01 2012-12-20 Ablynx N.V. Von willebrand factor specific binding agents and uses thereof
HRP20251657T1 (en) 2009-12-14 2026-02-13 Ablynx N.V. SINGLE VARIABLE DOMAIN IMMUNOGLOBULIN ANTIBODIES AGAINST OX40L, CONSTRUCTS AND THEIR THERAPEUTIC USE
WO2011083141A2 (en) 2010-01-08 2011-07-14 Ablynx Nv Method for generation of immunoglobulin sequences by using lipoprotein particles
TWI513466B (en) 2010-01-20 2015-12-21 Boehringer Ingelheim Int Anticoagulant antidotes
CN102781959A (en) 2010-02-05 2012-11-14 埃博灵克斯股份有限公司 Peptides capable of binding to serum albumin and compounds, constructs and polypeptides comprising the same
US9120855B2 (en) 2010-02-10 2015-09-01 Novartis Ag Biologic compounds directed against death receptor 5
ES2738114T3 (en) 2010-02-11 2020-01-20 Ablynx Nv Methods and compositions for aerosol preparation
EP2533814A2 (en) 2010-02-11 2012-12-19 Ablynx N.V. Delivery of immunoglobulin variable domains and constructs thereof
US8337845B2 (en) 2010-03-03 2012-12-25 Boehringer Ingelheim International Gmbh A-beta binding polypeptides
EP2552962B1 (en) 2010-03-26 2016-03-23 Ablynx N.V. Immunoglobulin single variable domains directed against cxcr7
CA2794551A1 (en) 2010-03-26 2011-09-29 Westfaelische Wilhelms-Universitaet Muenster Substitute therapy for glucocorticoids
US9101674B2 (en) 2010-03-29 2015-08-11 Vib Vzw Targeting and in vivo imaging of tumor-associated macrophages
US9556273B2 (en) 2010-03-29 2017-01-31 Vib Vzw Anti-macrophage mannose receptor single variable domains for targeting and in vivo imaging of tumor-associated macrophages
CA2798432A1 (en) 2010-05-06 2011-11-10 Novartis Ag Compositions and methods of use for therapeutic low density lipoprotein -related protein 6 (lrp6) antibodies
CN102939538A (en) 2010-05-07 2013-02-20 霍夫曼-拉罗奇有限公司 Diagnostic method for the detection of cells ex vivo
MX2012013490A (en) 2010-05-20 2013-01-29 Ablynx Nv BIOLOGICAL MATERIALS RELATED TO HER3.
WO2011161263A1 (en) 2010-06-25 2011-12-29 Ablynx Nv Pharmaceutical compositions for cutaneous administration
WO2012007880A2 (en) 2010-07-16 2012-01-19 Ablynx Nv Modified single domain antigen binding molecules and uses thereof
MX345300B (en) 2010-07-30 2017-01-24 Novartis Ag * Fibronectin cradle molecules and libraries thereof.
SG187908A1 (en) 2010-08-20 2013-03-28 Novartis Ag Antibodies for epidermal growth factor receptor 3 (her3)
US20120225081A1 (en) 2010-09-03 2012-09-06 Boehringer Ingelheim International Gmbh Vegf-binding molecules
US20120244141A1 (en) 2010-09-28 2012-09-27 Boehringer Ingelheim International Gmbh Stratification of cancer patients for susceptibility to therapy with PTK2 inhibitors
US11644471B2 (en) 2010-09-30 2023-05-09 Ablynx N.V. Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains
EP2621953B1 (en) 2010-09-30 2017-04-05 Ablynx N.V. Biological materials related to c-met
ES2660895T3 (en) 2010-10-29 2018-03-26 Ablynx N.V. Method for the production of individual variable domains of immunoglobulin
EA037063B1 (en) 2010-11-08 2021-02-01 Аблинкс Н.В. Cxcr2 binding polypeptides
US9598499B2 (en) * 2010-12-30 2017-03-21 Institut National De La Santé Et De La Recherche Médicale (Inserm) Antigen binding formats for use in therapeutic treatments or diagnostic assays
PE20140982A1 (en) 2011-01-06 2014-08-22 Glaxo Group Ltd LINKS THAT JOIN THE TRANSFORMANT GROWTH FACTOR-BETA RECEPTOR II
EP2683413A1 (en) 2011-03-07 2014-01-15 F.Hoffmann-La Roche Ag In vivo selection of therapeutically active antibodies
US20140099264A1 (en) 2011-03-07 2014-04-10 F. Hoffman-La Roche Ag Means and methods for in vivo testing of therapeutic antibodies
EP2691418A1 (en) 2011-03-28 2014-02-05 Ablynx N.V. Bispecific anti-cxcr7 immunoglobulin single variable domains
US9468679B2 (en) 2011-03-28 2016-10-18 Ablynx N.V. Method for producing solid formulations comprising immunoglobulin single variable domains
PE20140964A1 (en) 2011-03-30 2014-08-17 Boehringer Ingelheim Int ANTICOAGULANT ANTIDOTES
CN106039306A (en) 2011-03-30 2016-10-26 埃博灵克斯股份有限公司 Methods of treating immune disorders with single domain antibodies against TNF-alpha
US20130078247A1 (en) * 2011-04-01 2013-03-28 Boehringer Ingelheim International Gmbh Bispecific binding molecules binding to dii4 and ang2
US9527925B2 (en) 2011-04-01 2016-12-27 Boehringer Ingelheim International Gmbh Bispecific binding molecules binding to VEGF and ANG2
UA117218C2 (en) 2011-05-05 2018-07-10 Мерк Патент Гмбх POLYPEPTIDE AGAINST IL-17A, IL-17F AND / OR IL17-A / F
US9534039B2 (en) 2011-05-09 2017-01-03 Ablynx N.V. Method for the production of immunoglobulin single variable domains
KR102072250B1 (en) 2011-05-27 2020-03-02 아블린쓰 엔.브이. Inhibition of bone resorption with rankl binding peptides
US9580480B2 (en) 2011-05-31 2017-02-28 Massachusetts Institute Of Technology Cell-directed synthesis of multifunctional nanopatterns and nanomaterials
EP4350345A3 (en) 2011-06-23 2024-07-24 Ablynx N.V. Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobin single variable domains
JP2014525736A (en) * 2011-06-23 2014-10-02 アブリンクス エン．ヴェー． Immunoglobulin single variable domain for IgE
EP2944654A1 (en) 2011-06-23 2015-11-18 Ablynx N.V. Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobin single variable domains
EP3466972B1 (en) 2011-06-23 2026-03-04 Ablynx NV Serum albumin binding proteins
HUE031828T2 (en) 2011-06-23 2017-08-28 Ablynx Nv Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains
AU2012311286B2 (en) 2011-09-19 2018-07-26 Kymab Limited Antibodies, variable domains and chains tailored for human use
EP2747782B1 (en) 2011-09-23 2018-01-17 Ablynx NV Prolonged inhibition of interleukin-6 mediated signaling
WO2013045916A1 (en) 2011-09-26 2013-04-04 Kymab Limited Chimaeric surrogate light chains (slc) comprising human vpreb
CN103889451B (en) 2011-09-30 2016-06-29 埃博灵克斯股份有限公司 Biological substances associated with C-Met
US9346884B2 (en) 2011-09-30 2016-05-24 Ablynx N.V. Biological materials related to c-Met
GB2496375A (en) 2011-10-28 2013-05-15 Kymab Ltd A non-human assay vertebrate comprising human antibody loci and human epitope knock-in, and uses thereof
KR20140091031A (en) 2011-11-04 2014-07-18 노파르티스 아게 Low density lipoprotein-related protein 6 (lrp6) - half life extender constructs
GB201122047D0 (en) 2011-12-21 2012-02-01 Kymab Ltd Transgenic animals
JP2015500829A (en) 2011-12-05 2015-01-08 ノバルティス アーゲー HER3 antibody against domain II of epidermal growth factor receptor 3 (HER3)
KR102083957B1 (en) 2011-12-05 2020-03-04 노파르티스 아게 Antibodies for epidermal growth factor receptor 3 (her3)
US20140363434A1 (en) * 2012-01-06 2014-12-11 Complix Nv Binding agents to intracellular target molecules
CA2865464C (en) 2012-03-30 2022-07-12 Boehringer Ingelheim International Gmbh Ang2-binding molecules
US9328174B2 (en) 2012-05-09 2016-05-03 Novartis Ag Chemokine receptor binding polypeptides
KR20220051197A (en) 2012-05-17 2022-04-26 익스텐드 바이오사이언시즈, 인크. Carriers for improved drug delivery
AU2013265665B2 (en) 2012-05-24 2017-10-26 Vib Vzw Anti-macrophage mannose receptor single variable domains for targeting and in vivo imaging of tumor-associated macrophages
US11339208B1 (en) 2012-05-31 2022-05-24 United States Of America As Represented By The Secretary Of The Air Force Camelidae single-domain antibodies against Yersinia pestis and methods of use
WO2014030780A1 (en) * 2012-08-22 2014-02-27 Mogam Biotechnology Research Institute Screening and engineering method of super-stable immunoglobulin variable domains and their uses
EP2977386A1 (en) * 2012-08-31 2016-01-27 Argen-X Nv Method for producing antibody molecules having inter-species, intra-target cross-reactivity
US20140161796A1 (en) * 2012-09-13 2014-06-12 Andreas Loew Single chain proteins with c-terminal modifications
RU2530553C2 (en) * 2012-11-07 2014-10-10 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Recombinant single-domain antibody able to bind human tumour necrosis factor specifically and its derivatives
WO2014087010A1 (en) 2012-12-07 2014-06-12 Ablynx N.V. IMPROVED POLYPEPTIDES DIRECTED AGAINST IgE
EP2752426A1 (en) 2013-01-03 2014-07-09 Covagen AG Human serum albumin binding compounds and fusion proteins thereof
WO2014111550A1 (en) 2013-01-17 2014-07-24 Glaxosmithkline Intellectual Property Development Limited Modified anti-serum albumin binding proteins
CN112858672A (en) 2013-01-30 2021-05-28 弗拉芒区生物技术研究所 Novel chimeric polypeptides for screening and drug discovery purposes
WO2014120916A1 (en) 2013-02-01 2014-08-07 Bristol-Myers Squibb Company Pegylated domain antibodies monovalent for cd28 binding and methods of use
PL2953973T3 (en) 2013-02-05 2020-01-31 Vib Vzw Muscarinic acetylcholine receptor binding agents and uses thereof
JP2016514130A (en) 2013-03-14 2016-05-19 ノバルティス アーゲー Antibody against Notch3
WO2014140376A1 (en) 2013-03-15 2014-09-18 Vib Vzw Anti-macrophage mannose receptor single variable domains for use in cardiovascular diseases
JP6967347B2 (en) 2013-04-29 2021-11-17 バイオタリス・エン・フェー Agricultural chemical composition containing an antibody that binds to sphingolipids
NL1040254C2 (en) 2013-05-17 2014-11-24 Ablynx Nv Stable formulations of immunoglobulin single variable domains and uses thereof.
JP6449272B2 (en) * 2013-07-08 2019-01-09 ナンジン レジェンド バイオテック カンパニー，リミテッドＮａｎｊｉｎｇ Ｌｅｇｅｎｄ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． Compositions and methods for increasing the serum half-life of proteins
CN103454413B (en) * 2013-09-04 2015-03-11 吉日木图 Bactrian camel specific antibody preparation and immunodetection method
EP2883883A1 (en) 2013-12-16 2015-06-17 Cardio3 Biosciences S.A. Therapeutic targets and agents useful in treating ischemia reperfusion injury
EP3087101B1 (en) 2013-12-20 2024-06-05 Novartis AG Regulatable chimeric antigen receptor
US20170081411A1 (en) 2014-03-15 2017-03-23 Novartis Ag Regulatable chimeric antigen receptor
US10501540B2 (en) * 2014-04-10 2019-12-10 Lava Therapeutics B.V. Immunoglobulins binding human Vγ9 Vδ2 T cell receptors
SG11201609162SA (en) 2014-05-16 2016-12-29 Ablynx Nv Improved immunoglobulin variable domains
AU2015261467B2 (en) 2014-05-16 2020-12-17 Ablynx Nv Methods for detecting and/or measuring anti-drug antibodies, in particular treatment-emergent anti-drug antibodies
IL318433A (en) 2014-05-16 2025-03-01 Ablynx Nv Improved immunoglobulin variable domains
NL2013007B1 (en) 2014-06-16 2016-07-05 Ablynx Nv Methods of treating TTP with immunoglobulin single variable domains and uses thereof.
NL2013661B1 (en) 2014-10-21 2016-10-05 Ablynx Nv KV1.3 Binding immunoglobulins.
US11542488B2 (en) 2014-07-21 2023-01-03 Novartis Ag Sortase synthesized chimeric antigen receptors
WO2016012363A1 (en) 2014-07-22 2016-01-28 Vib Vzw Methods to select for agents that stabilize protein complexes
JP6289733B2 (en) 2014-07-29 2018-03-07 フレイエ ユニヴェルシテイト ブリュッセルＶｒｉｊｅ Ｕｎｉｖｅｒｓｉｔｅｉｔ Ｂｒｕｓｓｅｌ Radiolabeled antibody fragments for use in the prevention and / or treatment of cancer
US20180036442A1 (en) 2014-07-29 2018-02-08 Vrije Universiteit Brussel Radio-labelled antibody fragments for use in the prognosis, diagnosis of cancer as well as for the prediction of cancer therapy response
US20170209492A1 (en) 2014-07-31 2017-07-27 Novartis Ag Subset-optimized chimeric antigen receptor-containing t-cells
US11111284B2 (en) 2014-08-21 2021-09-07 The General Hospital Corporation Tumor necrosis factor superfamily and TNF-like ligand muteins and methods of preparing
WO2016065052A1 (en) 2014-10-22 2016-04-28 Extend Biosciences, Inc. Insulin vitamin d conjugates
US9585934B2 (en) 2014-10-22 2017-03-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US9789197B2 (en) 2014-10-22 2017-10-17 Extend Biosciences, Inc. RNAi vitamin D conjugates
US20170267784A1 (en) 2014-10-23 2017-09-21 Singh Molecular Medicine, Llc Single domain antibodies directed against intracellular antigens
DK3590962T3 (en) 2014-10-23 2021-12-13 Singh Molecular Medicine Llc SINGLE DOMAIN ANTIBODIES TARGETED TO INTRACELLULAR ANTIGENS
AR102550A1 (en) 2014-11-05 2017-03-08 Agrosavfe Nv TRANSGENIC PLANT THAT INCLUDES A POLINUCLEOTIDE THAT CODIFIES A VARIABLE DOMAIN OF HEAVY CHAIN ANTIBODY
WO2016094602A1 (en) 2014-12-10 2016-06-16 Tufts University Vhh based binding antibodies for anthrax and botulinum toxins and methods of making and using therefor
CN107406497A (en) 2014-12-19 2017-11-28 埃博灵克斯股份有限公司 The nano antibody dimer of cysteine connection
AU2016212777B2 (en) 2015-01-27 2021-11-18 LAVA Therapeutics N.V. Single domain antibodies targeting CD1d
PT3294768T (en) 2015-05-13 2019-11-05 Ablynx Nv T cell recruiting polypeptides based on tcr alpha/beta reactivity
US11046767B2 (en) 2015-05-13 2021-06-29 Ablynx N.V. T cell recruiting polypeptides based on CD3 reactivity
BR112017024757A2 (en) 2015-05-18 2018-11-13 TCR2 Therapeutics Inc. compositions and methods for reprogramming tcr using fusion proteins
WO2017013026A1 (en) 2015-07-17 2017-01-26 Vrije Universiteit Brussel Radiolabelled antibody fragments for use in treating cancer
BR102016018074A2 (en) 2015-08-07 2021-11-16 ALX Oncology Inc. SIRP-ALFA VARIANT CONSTRUCTION, ITS METHOD OF PREPARATION AND USES, NUCLEIC ACID MOLECULE, VECTOR, HOST CELL, AND PHARMACEUTICAL COMPOSITION
WO2017027422A1 (en) 2015-08-07 2017-02-16 Alexo Therapeutics Inc. Constructs having a sirp-alpha domain or variant thereof
TWI746473B (en) 2015-11-02 2021-11-21 美商辛分子醫藥有限公司 Single domain antibodies directed against intracellular antigens
NO2768984T3 (en) 2015-11-12 2018-06-09
ES2905912T3 (en) 2015-11-13 2022-04-12 Ablynx Nv Enhanced serum albumin-binding immunoglobulin variable domains
US11077187B2 (en) * 2015-11-17 2021-08-03 Oklahoma Medical Research Foundation Epitope of optimized humanized monoclonal antibodies against activated protein C and uses thereof
EP3377525A2 (en) 2015-11-18 2018-09-26 Ablynx NV Improved serum albumin binders
MY189590A (en) 2015-11-18 2022-02-18 Merck Sharp & Dohme Ctla4 binders
CA3132021C (en) 2015-11-18 2024-03-12 Merck Sharp & Dohme Corp. Pd1 and/or lag3 binders
CA3003777A1 (en) 2015-11-18 2017-05-26 Merck Sharp & Dohme Corp. Pd1/ctla4 binders
CN108473561B (en) 2015-11-27 2022-12-16 埃博灵克斯股份有限公司 Polypeptide inhibiting CD40L
IL298686A (en) * 2016-01-11 2023-01-01 Inhibrx Inc Multispecific and multivalent ox40-binding fusion proteins
WO2017124001A2 (en) * 2016-01-14 2017-07-20 Memorial Sloan-Kettering Cancer Center T cell receptor-like antibodies specific for foxp3-derived peptides
EP3408299A1 (en) 2016-01-26 2018-12-05 INSERM - Institut National de la Santé et de la Recherche Médicale Anti-vwf d'd3 single-domain antibodies and polypeptides comprising thereof
CN116769054A (en) 2016-02-05 2023-09-19 奥里尼斯生物科学私人有限公司 Bispecific signaling agents and uses thereof
EP4276114A3 (en) 2016-03-07 2024-02-21 Vib Vzw Cd20 binding single domain antibodies
WO2017182605A1 (en) 2016-04-22 2017-10-26 Université Libre de Bruxelles A new biomarker expressed in pancreatic beta cells useful in imaging or targeting beta cells
US11243214B2 (en) 2016-04-22 2022-02-08 Université Libre de Bruxelles Biomarker expressed in pancreatic beta cells useful in imaging or targeting beta cells
CA3022697A1 (en) 2016-05-02 2017-11-09 Ablynx Nv Treatment of rsv infection
US11753463B2 (en) 2016-05-13 2023-09-12 Orionis Biosciences BV Therapeutic targeting of non-cellular structures
UA127200C2 (en) 2016-05-18 2023-06-07 Бьорінгер Інгельхайм Інтернаціональ Гмбх Anti pd-1 and anti-lag3 antibodies for cancer treatment
CN109313183B (en) 2016-06-23 2022-10-21 埃博灵克斯股份有限公司 Improved pharmacokinetic assay for immunoglobulin single variable domains
WO2018007442A1 (en) 2016-07-06 2018-01-11 Ablynx N.V. Treatment of il-6r related diseases
WO2018026953A1 (en) 2016-08-02 2018-02-08 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using fusion proteins
WO2018029182A1 (en) 2016-08-08 2018-02-15 Ablynx N.V. Il-6r single variable domain antibodies for treatment of il-6r related diseases
RU2762835C2 (en) 2016-08-24 2021-12-23 Тенеобио, Инк. Transgenic animals, other than humans, producing modified antibodies containing only heavy chains
CN106267191A (en) * 2016-09-03 2017-01-04 山西纳安生物科技有限公司 Nano antibody biological medicament Percutaneously administrable preparation system and preparation method and application
CN106265480A (en) * 2016-09-03 2017-01-04 山西纳安生物科技有限公司 Nano antibody vaginal drug delivery systems and preparation method and application
US11098113B2 (en) 2016-09-15 2021-08-24 Vib Vzw Immunoglobulin single variable domains directed against macrophage migration inhibitory factor
MX2019003899A (en) 2016-10-07 2019-08-14 Tcr2 Therapeutics Inc Compositions and methods for t-cell receptors reprogramming using fusion proteins.
IL317416A (en) 2016-11-16 2025-02-01 Ablynx Nv T cell recruiting polypeptides capable of binding cd123 and tcr alpha/beta
AU2017363311A1 (en) 2016-11-22 2019-06-13 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins
WO2018099968A1 (en) 2016-11-29 2018-06-07 Ablynx N.V. Treatment of infection by respiratory syncytial virus (rsv)
EP3551656A1 (en) 2016-12-07 2019-10-16 Ablynx NV Improved serum albumin binding immunoglobulin single variable domains
EP3360898A1 (en) 2017-02-14 2018-08-15 Boehringer Ingelheim International GmbH Bispecific anti-tnf-related apoptosis-inducing ligand receptor 2 and anti-cadherin 17 binding molecules for the treatment of cancer
CN107674122A (en) * 2016-12-28 2018-02-09 天津天锐生物科技有限公司 A kind of single domain antibody for identifying human serum albumins
CN117285623A (en) 2017-01-17 2023-12-26 埃博灵克斯股份有限公司 Improved serum albumin conjugate
DK3571224T3 (en) 2017-01-17 2024-11-11 Ablynx Nv IMPROVED SERUM ALBUMIN BINDERS
GB201701404D0 (en) * 2017-01-27 2017-03-15 Micropharm Ltd Therapies for treating inflammatory disorders
MX2019009255A (en) 2017-02-06 2019-11-05 Orionis Biosciences Nv Targeted chimeric proteins and uses thereof.
BR112019017853A2 (en) 2017-02-28 2021-04-27 Vib Vzw MEANS AND METHODS FOR ORAL RELEASE OF PROTEIN
JP7165717B2 (en) 2017-03-15 2022-11-04 パンディオン・オペレーションズ・インコーポレイテッド target immune tolerance
EP3600453A1 (en) 2017-03-30 2020-02-05 Duke University Radiolabeled biomolecules and their use
AU2018241286B2 (en) 2017-03-31 2025-03-13 Ablynx N.V. Improved immunogenicity assays
EP3612648A1 (en) 2017-04-18 2020-02-26 Université Libre de Bruxelles Biomarkers and targets for proliferative diseases
CA3062238A1 (en) 2017-05-11 2018-11-15 Vib Vzw Glycosylation of variable immunoglobulin domains
AU2018273914A1 (en) 2017-05-24 2019-11-14 Pandion Operations, Inc. Targeted immunotolerance
CN110997001A (en) 2017-06-02 2020-04-10 默克专利股份有限公司 MMP13 binding to immunoglobulins
MX2019014504A (en) 2017-06-02 2020-07-20 Merck Patent Gmbh AGRECAN-BINDING IMMUNOGLOBULINS.
CN111032695B (en) 2017-06-02 2024-06-25 默克专利股份有限公司 ADAMTS-binding immunoglobulin
MX2019014397A (en) 2017-06-02 2020-02-10 Merck Patent Gmbh Polypeptides binding adamts5, mmp13 and aggrecan.
CN107356572A (en) * 2017-06-28 2017-11-17 成都理工大学 The FRET method of carrier is used as by the use of nucleic acid
GB201710973D0 (en) 2017-07-07 2017-08-23 Avacta Life Sciences Ltd Scaffold proteins
CN108570095B (en) * 2017-07-10 2019-12-31 昆明医科大学第一附属医院 A Polypeptide SITE2 Promoting Platelet Aggregation
CN111201246B (en) 2017-07-11 2023-10-27 亚力兄制药公司 Polypeptides that bind complement component C5 or serum albumin and their fusion proteins
CA3070253A1 (en) 2017-07-19 2019-01-24 Vib Vzw Serum albumin binding agents
EP4635987A3 (en) 2017-10-31 2026-01-14 Vib Vzw Novel antigen-binding chimeric proteins and methods and uses thereof
CN111386285B (en) * 2017-11-15 2024-08-27 诺和诺德股份有限公司 Factor X binders that enhance FX activation
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
USRE50550E1 (en) 2017-12-06 2025-08-26 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10174091B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
CN109970860A (en) * 2017-12-27 2019-07-05 信达生物制药(苏州)有限公司 Three chain antibodies, Its Preparation Method And Use
WO2019148089A1 (en) 2018-01-26 2019-08-01 Orionis Biosciences Inc. Xcr1 binding agents and uses thereof
CN112074267B (en) 2018-02-05 2024-06-28 奥里尼斯生物科学公司股份有限公司 Fibroblast binding agent and use thereof
CN111670202A (en) 2018-02-06 2020-09-15 埃博灵克斯股份有限公司 Methods of treating initial episodes of TTP with immunoglobulin single variable domains
WO2019155041A1 (en) 2018-02-12 2019-08-15 Vib Vzw Gβγ COMPLEX ANTIBODIES AND USES THEREOF
TW202000238A (en) 2018-02-26 2020-01-01 比利時商艾伯林克斯公司 Improved nucleotide sequences encoding peptide linkers
EP3758742A1 (en) 2018-03-01 2021-01-06 Vrije Universiteit Brussel Human pd-l1-binding immunoglobulins
JP7390729B2 (en) 2018-03-23 2023-12-04 ユニヴェルシテ リブル ドゥ ブリュッセル Wnt signaling agonist molecules
MX2020010091A (en) * 2018-03-27 2021-01-15 Umc Utrecht Holding Bv Targeted thrombolysis for treatment of microvascular thrombosis.
US20210130430A1 (en) 2018-05-14 2021-05-06 Werewolf Therapeutics, Inc. Activatable cytokine polypeptides and methods of use thereof
CN113840832A (en) 2018-05-14 2021-12-24 狼人治疗公司 Activatable interleukin-2 polypeptides and methods of using the same
WO2019222294A1 (en) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Activatable cytokine polypeptides and methods of use thereof
EP3569618A1 (en) 2018-05-19 2019-11-20 Boehringer Ingelheim International GmbH Antagonizing cd73 antibody
MX2020013248A (en) * 2018-06-05 2021-04-13 Novobind Livestock Therapeutics Inc Antibodies against aquaculture disease-causing agents and uses thereof.
JP7122672B2 (en) * 2018-06-08 2022-08-22 パナソニックＩｐマネジメント株式会社 VHH antibodies
EP3807321A4 (en) 2018-06-18 2022-03-02 Anwita Biosciences, Inc. ANTI-MESOTHELIN CONSTRUCTIONS AND ASSOCIATED USES
UA130214C2 (en) 2018-07-20 2025-12-24 Момента Фармасьютикалз, Інк. PHARMACEUTICAL COMPOSITION OF ANTIBODY AGAINST FCRN
JP2021530503A (en) * 2018-07-26 2021-11-11 ティーシーアール２ セラピューティクス インク． Compositions and Methods for Reprogramming TCRs Using Target-Specific Fusion Proteins
CN112601761A (en) 2018-08-13 2021-04-02 印希比股份有限公司 Polypeptides that bind OX40 and uses thereof
JP7190675B2 (en) * 2018-08-23 2022-12-16 パナソニックＩｐマネジメント株式会社 Antibody and complex that bind to norovirus, detection device and detection method using the same
BR112021005184A8 (en) 2018-09-19 2022-08-16 Lava Therapeutics B V DUAL-ACTING CD1D IMMUNOGLOBULIN
MX2021003543A (en) 2018-09-27 2021-06-23 Xilio Dev Inc Masked cytokine polypeptides.
EP3636657A1 (en) 2018-10-08 2020-04-15 Ablynx N.V. Chromatography-free antibody purification method
CA3118892A1 (en) 2018-11-08 2020-05-14 Orionis Biosciences, Inc. Modulation of dendritic cell lineages
CA3118819A1 (en) * 2018-11-13 2020-05-22 Novobind Livestock Therapeutics Inc. Antibodies against disease causing agents of canines and felines and uses thereof
GB201818477D0 (en) 2018-11-13 2018-12-26 Emstopa Ltd Tissue plasminogen activator antibodies and method of use thereof
WO2020114616A1 (en) * 2018-12-07 2020-06-11 Tillotts Pharma Ag Topical treatment of immune checkpoint inhibitor induced diarrhoea, colitis or enterocolitis using antibodies and fragments thereof
CN112830967B (en) * 2019-01-10 2022-06-24 瑞阳(苏州)生物科技有限公司 Affinity chromatography purification method of anti-HSA single domain antibody and fusion protein thereof
JP7773372B2 (en) 2019-03-28 2025-11-19 オリオニス バイオサイエンシズ，インコーポレイテッド Fibroblast activation protein binding substances and uses thereof
CN114041057A (en) 2019-04-29 2022-02-11 康福治疗有限公司 Chimeric proteins and methods for screening compounds and ligands that bind to GPCRs
US20220289837A1 (en) 2019-04-30 2022-09-15 Vib Vzw Cystic Fibrosis Transmembrane Conductance Regulator Stabilizing Agents
KR20220023988A (en) 2019-05-14 2022-03-03 웨어울프 세라퓨틱스, 인크. Separation moieties and methods of use thereof
CA3141327A1 (en) 2019-05-20 2020-11-26 Pandion Operations, Inc. Madcam targeted immunotolerance
WO2020239934A1 (en) 2019-05-28 2020-12-03 Vib Vzw Cd8+ t-cells lacking plexins and their application in cancer treatment
EP3976650A1 (en) 2019-05-28 2022-04-06 Vib Vzw Cancer treatment by targeting plexins in the immune compartment
KR20220044904A (en) 2019-05-31 2022-04-12 알렉소 온콜로지 인크. How to treat cancer with SIRP alpha FC fusion in combination with immune checkpoint inhibitors
CA3138805A1 (en) 2019-06-04 2020-12-10 Johannes Schilling Designed ankyrin repeat domain with improved stability
CN110624105B (en) * 2019-09-24 2021-06-11 苏州大学 Sequences of structurally sensitive polypeptide antigens of von Willebrand factor
CN115052884B (en) * 2019-09-27 2026-03-17 北京烁星生物医药科技有限公司 Monospecific and multispecific antibodies
TW202128775A (en) 2019-10-16 2021-08-01 英商阿法克塔生命科學有限公司 Pd-l1 inhibitor - tgfβ inhibitor bispecific drug moieties
EP4048703A1 (en) 2019-10-21 2022-08-31 Vib Vzw Nanodisc-specific antigen-binding chimeric proteins
IL292879B2 (en) 2019-11-11 2025-08-01 Ibi Ag Innovative Bio Insecticides Ltd Insect control nanobodies and uses thereof
CN112851805B (en) * 2019-11-12 2025-09-09 四川科伦博泰生物医药股份有限公司 Protein combined with serum albumin and application thereof
BR112022009110A2 (en) 2019-11-14 2022-07-26 Werewolf Therapeutics Inc ACTIVABLE CYTOKINE POLYPEPTIDES AND METHODS OF USE THEREOF
EP4065603A1 (en) 2019-11-27 2022-10-05 Vib Vzw Positive allosteric modulators of the calcium-sensing receptor
CN114845710A (en) 2019-11-27 2022-08-02 Alx肿瘤生物技术公司 Combination therapy for the treatment of cancer
MX2022006881A (en) 2019-12-06 2022-07-11 Ablynx Nv POLYPEPTIDES COMPRISING SINGLE VARIABLE DOMAINS OF IMMUNOGLOBULIN TARGETTING TNF ALFA AND OX40L.
JP7657797B2 (en) 2019-12-06 2025-04-07 アブリンクス エン．ヴェー． Polypeptides Comprising Immunoglobulin Single Variable Domains That Target TNFα and IL-23 - Patent application
IL293548A (en) 2019-12-09 2022-08-01 Ablynx Nv Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp
GB201918279D0 (en) 2019-12-12 2020-01-29 Vib Vzw Glycosylated single chain immunoglobulin domains
EP4077372A1 (en) 2019-12-20 2022-10-26 Vib Vzw Nanobody exchange chromatography
GB201919294D0 (en) * 2019-12-24 2020-02-05 Eco Animal Health Ltd Antibodies or binding proteins
WO2021140205A1 (en) 2020-01-10 2021-07-15 Confo Therapeutics N.V. Methods for generating antibodies and antibody fragments and libraries comprising same
WO2021156490A2 (en) 2020-02-06 2021-08-12 Vib Vzw Corona virus binders
WO2021168079A1 (en) 2020-02-21 2021-08-26 Pandion Operations, Inc. Tissue targeted immunotolerance with a cd39 effector
JP7814714B2 (en) 2020-02-25 2026-02-17 ブイアイビー ブイゼットダブリュ Allosteric regulator of leucine-rich repeat kinase 2
CA3171066A1 (en) 2020-03-11 2021-09-16 Saar GILL Methods and composition for gene delivery using an engineered viral particle
EP4125379B1 (en) 2020-03-31 2024-08-21 Biotalys NV Anti-fungal polypeptides
CN113461824A (en) 2020-03-31 2021-10-01 普米斯生物技术(珠海)有限公司 Platform for constructing multispecific antibody
CN113527488B (en) 2020-04-22 2026-04-17 迈威(上海)生物科技股份有限公司 A monovariable domain antibody targeting human programmed death-ligand 1 (PD-L1) and its derivatives
WO2021229104A1 (en) 2020-05-15 2021-11-18 Université de Liège Anti-cd38 single-domain antibodies in disease monitoring and treatment
CA3180665A1 (en) 2020-05-19 2021-11-25 Boehringer Ingelheim International Gmbh Binding molecules for the treatment of cancer
GB202101299D0 (en) 2020-06-09 2021-03-17 Avacta Life Sciences Ltd Diagnostic polypetides and methods
WO2024133935A1 (en) 2022-12-23 2024-06-27 Ablynx Nv Protein-based conjugation carriers
WO2021258160A1 (en) * 2020-06-26 2021-12-30 Monash University Anti-vwf antibodies and uses thereof
WO2022003156A1 (en) 2020-07-02 2022-01-06 Oncurious Nv Ccr8 non-blocking binders
US12077576B2 (en) 2020-07-02 2024-09-03 Trustees Of Tufts College VHH polypeptides that bind to Clostridium difficile toxin b and methods of use thereof
IL299748A (en) 2020-07-08 2023-03-01 Lava Therapeutics N V Antibodies that bind psma and gamma-delta t cell receptors
WO2022023583A1 (en) 2020-07-31 2022-02-03 Biotalys NV Expression host
WO2022063947A1 (en) 2020-09-24 2022-03-31 Vib Vzw Combination of p2y6 inhibitors and immune checkpoint inhibitors
WO2022063957A1 (en) 2020-09-24 2022-03-31 Vib Vzw Biomarker for anti-tumor therapy
WO2022063984A1 (en) 2020-09-25 2022-03-31 Ablynx Nv Polypeptides comprising immunoglobulin single variable domains targeting il-13 and ox40l
JP7633397B2 (en) 2020-10-21 2025-02-19 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング Bispecific anti-VEGF and anti-TrkB binding molecules for the treatment of ocular diseases - Patents.com
WO2022098745A1 (en) 2020-11-03 2022-05-12 Indi Molecular, Inc. Compositions, delivery systems, and methods useful in tumor therapy
WO2022098743A1 (en) 2020-11-03 2022-05-12 Indi Molecular, Inc. Compositions, imaging, and therapeutic methods targeting folate receptor 1 (folr1)
JP2023553323A (en) 2020-11-25 2023-12-21 エクシリオ デベロップメント， インコーポレイテッド Tumor-specific cleavable linker
EP4255929A2 (en) 2020-12-02 2023-10-11 Vib Vzw An ltbr agonist in combination therapy against cancer
WO2022117569A1 (en) 2020-12-02 2022-06-09 Oncurious Nv A ccr8 antagonist antibody in combination with a lymphotoxin beta receptor agonist antibody in therapy against cancer
JP7842101B2 (en) 2020-12-18 2026-04-07 アブリンクス エン．ヴェー． polypeptide containing a single variable immunoglobulin domain that targets IL-6 and TNF-α
TW202241947A (en) 2020-12-18 2022-11-01 比利時商艾伯霖克斯公司 Polypeptides comprising immunoglobulin single variable domains targeting glypican-3 and t cell receptor
CA3203141A1 (en) 2020-12-18 2022-06-23 Ablynx Nv T cell recruiting polypeptides based on tcr alpha/beta reactivity
GB202020502D0 (en) 2020-12-23 2021-02-03 Vib Vzw Antibody composistion for treatment of corona virus infection
EP4267617A1 (en) 2020-12-24 2023-11-01 Vib Vzw Human ccr8 binders
EP4267621A1 (en) 2020-12-24 2023-11-01 Vib Vzw Murine cross-reactive human ccr8 binders
US20240052044A1 (en) 2020-12-24 2024-02-15 Vib Vzw Non-blocking human ccr8 binders
CN117794566A (en) 2021-02-05 2024-03-29 Vib研究所 Sabei virus binding agent
CA3207548A1 (en) 2021-02-05 2022-08-11 Xavier Saelens Sarbecovirus binders
EP4294407A1 (en) 2021-02-17 2023-12-27 Vib Vzw Inhibition of slc4a4 in the treatment of cancer
IL305318A (en) 2021-02-19 2023-10-01 Vib Vzw Cation-independent mannose-6-phosphate receptor binders
WO2022199804A1 (en) 2021-03-24 2022-09-29 Vib Vzw Nek6 inhibition to treat als and ftd
WO2022234003A1 (en) 2021-05-07 2022-11-10 Avacta Life Sciences Limited Cd33 binding polypeptides with stefin a protein
BR112023022774A2 (en) 2021-05-13 2024-01-02 Alx Oncology Inc COMBINATION THERAPIES FOR CANCER TREATMENT
US20240261446A1 (en) 2021-05-17 2024-08-08 Université de Liège Anti-cd38 single domain antibodies in disease monitoring and treatment
TW202317625A (en) 2021-06-17 2023-05-01 德商百靈佳殷格翰國際股份有限公司 Novel tri-specific binding molecules
WO2022268993A1 (en) 2021-06-23 2022-12-29 Vib Vzw Means and methods for selection of specific binders
US20240343811A1 (en) 2021-06-29 2024-10-17 Shandong Simcere Biopharmacutical Co., Ltd. Cd16 antibody and use thereof
WO2023016828A2 (en) 2021-07-30 2023-02-16 Vib Vzw Cation-independent mannose-6-phosphate receptor binders for targeted protein degradation
EP4378954A4 (en) 2021-07-30 2025-05-21 Shandong Simcere Biopharmaceutical Co., Ltd. BISPECIFIC ANTI-PVRIG/ANTI-TIGIT ANTIBODY AND USE
WO2023057601A1 (en) 2021-10-06 2023-04-13 Biotalys NV Anti-fungal polypeptides
TW202334196A (en) 2021-10-07 2023-09-01 英商阿法克塔生命科學有限公司 Pd-l1 binding polypeptides
EP4412711A1 (en) 2021-10-07 2024-08-14 Avacta Life Sciences Limited Serum half-life extended pd-l1 binding polypeptides
CN118696058A (en) 2021-12-03 2024-09-24 山东先声生物制药有限公司 Anti-BCMA nanoantibodies and their applications
CN118715246A (en) * 2021-12-10 2024-09-27 李人浩 Polypeptides, variants and uses thereof that bind to the A1 domain or auto-inhibitory module of von Willebrand factor (VWF)
JP2024546914A (en) 2021-12-15 2024-12-26 インテリウス・バイオセラピューティクス・インコーポレイテッド Pseudotyped virus particles, compositions containing same, and uses thereof
AU2022409733A1 (en) 2021-12-17 2024-08-01 Ablynx Nv POLYPEPTIDES COMPRISING IMMUNOGLOBULIN SINGLE VARIABLE DOMAINS TARGETING TCRαβ, CD33 AND CD123
US20250145705A1 (en) 2021-12-31 2025-05-08 Shandong Simcere Biopharmaceutical Co., Ltd. Gprc5d antibody and application thereof
EP4460516A2 (en) * 2022-01-03 2024-11-13 Twist Bioscience Corporation Bispecific sars-cov-2 antibodies and methods of use
CA3248241A1 (en) 2022-01-12 2023-07-20 Vib Vzw Human ntcp binders for therapeutic use and liver-specific targeted delivery
EP4473108A1 (en) 2022-02-02 2024-12-11 Biotalys NV Methods for genome editing
EP4476250A1 (en) 2022-02-07 2024-12-18 Vib Vzw Engineered stabilizing aglycosylated fc-regions
WO2023153876A1 (en) 2022-02-10 2023-08-17 주식회사 아피셀테라퓨틱스 Stefin a protein variants specifically binding to cd40l, and uses thereof
US20250235534A1 (en) 2022-04-13 2025-07-24 Vib Vzw An LTBR Agonist In Combination Therapy Against Cancer
EP4519324A1 (en) 2022-05-02 2025-03-12 UMC Utrecht Holding B.V. Single domain antibodies for the detection of plasmin-cleaved vwf
WO2023218243A1 (en) 2022-05-12 2023-11-16 Avacta Life Sciences Limited Lag-3/pd-l1 binding fusion proteins
EP4526340A1 (en) 2022-05-18 2025-03-26 Vib Vzw Sarbecovirus spike s2 subunit binders
KR20250021313A (en) 2022-06-06 2025-02-12 산둥심시어 바이오파마슈티칼 씨오., 엘티디. Multispecific antibodies targeting BCMA, GPRC5D and T cells and their applications
CA3259417A1 (en) 2022-06-14 2023-12-21 Ablynx Nv Immunoglobulin single variable domains targeting t cell receptor
WO2024003873A1 (en) * 2022-06-30 2024-01-04 Intrexon Actobiotics Nv D/B/A Precigen Actobio Single variable domain antibodies against tumor necrosis factor-alpha
JP2025523630A (en) 2022-07-04 2025-07-23 ブイアイビー ブイゼットダブリュ Blood-cerebrospinal fluid barrier crossing antibodies
US12098400B2 (en) 2022-07-08 2024-09-24 Novo Nordisk A/S Highly potent ISVD compounds capable of substituting for FVIII(A)
EP4554677A1 (en) 2022-07-15 2025-05-21 Boehringer Ingelheim International GmbH Binding molecules for the treatment of cancer
CA3262768A1 (en) 2022-07-25 2024-02-01 Interius Biotherapeutics, Inc. Mutated polypeptides, compositions comprising the same, and uses thereof
KR20250042176A (en) 2022-07-27 2025-03-26 아블린쓰 A polypeptide that binds to a specific epitope of the neonatal FC receptor
EP4594348A1 (en) 2022-09-27 2025-08-06 Vib Vzw Antivirals against human parainfluenza virus
EP4593867A2 (en) 2022-09-30 2025-08-06 Extend Biosciences, Inc. Long-acting parathyroid hormone
WO2024083843A1 (en) 2022-10-18 2024-04-25 Confo Therapeutics N.V. Amino acid sequences directed against the melanocortin 4 receptor and polypeptides comprising the same for the treatment of mc4r-related diseases and disorders
KR20250102088A (en) 2022-11-09 2025-07-04 메르크 파텐트 게엠베하 Toll-like receptor 7 agonist as an immunostimulant for inducing innate antitumor immunity
WO2024105091A1 (en) 2022-11-15 2024-05-23 Imec Vzw Method and system for droplet manipulation
EP4634232A1 (en) * 2022-12-15 2025-10-22 Institut National de la Santé et de la Recherche Médicale Single-domain antibody targeting von wilebrand factor a3-domain
US20240200085A1 (en) 2022-12-15 2024-06-20 Aarhus Universitet Synthetic activation of multimeric transmembrane receptors
WO2024133937A1 (en) 2022-12-22 2024-06-27 Biotalys NV Methods for genome editing
WO2024141921A1 (en) * 2022-12-26 2024-07-04 National Institute Of Pharmaceutical Education And Research (Niper) Tumour necrosis factor alpha domain antibody
US20260026500A1 (en) 2022-12-29 2026-01-29 Biotalys NV Agrochemical compositions
EP4642233A1 (en) 2022-12-30 2025-11-05 Biotalys NV Agglomerate
WO2024141638A1 (en) 2022-12-30 2024-07-04 Biotalys NV Self-emulsifiable concentrate
EP4642916A2 (en) 2022-12-30 2025-11-05 Biotalys NV Secretion signals
CN120529914A (en) 2023-01-09 2025-08-22 奥德赛治疗股份有限公司 Anti-TNFR2 antigen binding protein and its use
EP4655323A1 (en) 2023-01-27 2025-12-03 Vrije Universiteit Brussel Cd8b-binding polypeptides
WO2024156888A1 (en) 2023-01-27 2024-08-02 Vib Vzw Cd163-binding conjugates
WO2024163894A1 (en) 2023-02-04 2024-08-08 Momenta Pharmaceuticals, Inc. Compositions and methods for treating hemolytic disease of the fetus and newborn
WO2024165710A1 (en) 2023-02-09 2024-08-15 Seni-Preps B.V. Immunoglobulin single variable domains that inhibit urease and use thereof
AR131869A1 (en) 2023-02-17 2025-05-07 Ablynx Nv POLYPEPTIDES THAT BIND TO THE NEONATAL Fc RECEPTOR
WO2024175787A1 (en) 2023-02-24 2024-08-29 Vrije Universiteit Brussel Anti-inflammatory pannexin 1 channel inhibitors
AU2024236537A1 (en) 2023-03-14 2025-09-04 Aarhus Universitet Genetically altered nfr5 receptor kinases
IL323035A (en) 2023-03-14 2025-10-01 Odyssey Therapeutics Inc Anti-cd25 antigen-binding proteins and uses thereof
FR3147278A1 (en) 2023-03-31 2024-10-04 Avacta Life Sciences Limited TNFR2-BINDING POLYPEPTIDES AND METHODS OF USE
EP4688825A1 (en) 2023-03-31 2026-02-11 AffyXell Therapeutics Co., Ltd. Tnfr2 binding polypeptides and methods of use
JP2026513540A (en) 2023-03-31 2026-04-28 オクタファルマ アクチェン ゲゼルシャフト FVIII-VWF fusion protein with improved pharmacokinetics
WO2024208816A1 (en) 2023-04-03 2024-10-10 Vib Vzw Blood-brain barrier crossing antibodies
AR132494A1 (en) 2023-04-24 2025-07-02 Merck Sharp & Dohme Llc TROP2 BINDERS AND CONJUGATES THEREOF
CN121511260A (en) 2023-05-11 2026-02-10 非营利性组织佛兰芒综合大学生物技术研究所 SLC4A4/NBCe1 inhibitors
CN121511255A (en) 2023-05-17 2026-02-10 奥德赛治疗股份有限公司 Modified single-domain antibodies
AU2024276787A1 (en) 2023-05-23 2026-01-15 Allygen Group Pd-l1 and trop-2 targeting conjugates comprising effector molecules and uses thereof
EP4731770A1 (en) 2023-06-22 2026-04-29 Ablynx NV Chimeric proteins for modulating cytokine receptor activity
WO2024261344A1 (en) 2023-06-23 2024-12-26 Vib Vzw Novel binders targeting the multi-drug resistant pathogen acinetobacter baumannii
KR20260032566A (en) 2023-06-29 2026-03-09 오디세이 테라퓨틱스, 인코포레이티드 Anti-TRAILR2 antigen-binding protein and uses thereof
EP4483951A1 (en) 2023-06-30 2025-01-01 Université de Liège Single-domain antibody for inhibition of neutrophil elastase activity
CN121443644A (en) 2023-07-05 2026-01-30 阿布林克斯有限公司 FcRn antagonists for the treatment of IgG-related diseases and disorders.
CN121925428A (en) 2023-07-30 2026-04-24 詹森生物科技公司 Molecules that bind mutant calreticulin and uses thereof
TW202521562A (en) 2023-08-11 2025-06-01 美商默沙東有限責任公司 Monovalent interleukin 12 (il-12) heterodimeric fc proteins
AR133725A1 (en) 2023-09-04 2025-10-29 Sanofi Sa POLYPEPTIDES FOR USE IN THE TREATMENT OF GLYPICAN-3 EXPRESSING TUMORS
TW202527982A (en) 2023-09-11 2025-07-16 美商默門塔醫藥公司 Pharmaceutical compositions of fcrn antibodies
EP4626931A1 (en) * 2023-09-17 2025-10-08 Ji Yan Biomedical Co., Ltd Recombinant antibodies and uses thereof
CN121986112A (en) 2023-09-22 2026-05-05 阿布林克斯有限公司 Divalent and multivalent albumin binders
NL2036011B1 (en) 2023-10-12 2025-04-30 Synapse Res Institute Molecules for reversing anti-coagulant activity of direct oral anticoagulants
WO2025080751A2 (en) 2023-10-13 2025-04-17 Odyssey Therapeutics, Inc. Anti-cdh17 antigen-binding proteins and uses thereof
WO2025093683A1 (en) 2023-11-03 2025-05-08 Neuvasq Biotechnologies Sa Wnt7 signaling agonists
WO2025099576A1 (en) 2023-11-06 2025-05-15 Momenta Pharmaceuticals, Inc. Compositions and methods for treating rheumatoid arthritis
WO2025099632A1 (en) 2023-11-08 2025-05-15 Sanofi Cd25 based lysosomal degrader and uses thereof
CN117229423B (en) * 2023-11-10 2024-02-06 北京科技大学 A polypeptide nanomaterial for binding collagen and its preparation method and application
US20250376509A1 (en) 2023-11-21 2025-12-11 Janssen Biotech, Inc. Methods for treatment of myeloproliferative neoplasms
WO2025109176A1 (en) 2023-11-22 2025-05-30 Exevir Bio Bv Optimized sarbecovirus spike s2 subunit binders and compositions comprising the same
AR134512A1 (en) 2023-12-01 2026-01-21 Ablynx Nv PRECISION-ACTIVATED POLYPEPTIDES
WO2025125577A1 (en) 2023-12-14 2025-06-19 Vib Vzw Antibodies against influenza b virus
TW202543670A (en) 2023-12-22 2025-11-16 比利時商艾伯霖克斯公司 Protein-based conjugation carriers for intranuclear delivery
AR134804A1 (en) 2023-12-22 2026-04-01 Ablynx Nv PROTEIN-BASED CARRIERS FOR SITE-SPECIFIC AMINE CONJUGATION
US12378306B2 (en) 2023-12-22 2025-08-05 Biotalys NV Anti-fungal VHH antibodies
WO2025149667A1 (en) 2024-01-12 2025-07-17 Pheon Therapeutics Ltd Antibody drug conjugates and uses thereof
WO2025154056A1 (en) 2024-01-21 2025-07-24 Ibi-Ag Innovative Bio Insecticides Ltd. Anti-insect cda nanobodies and uses thereof
WO2025154058A1 (en) 2024-01-21 2025-07-24 Ibi-Ag Innovative Bio Insecticides Ltd. Anti-insect hsp70 nanobodies and uses thereof
WO2025163617A1 (en) 2024-02-04 2025-08-07 Momenta Pharmaceuticals, Inc. Compositions and methods for treating sjögren's disease
WO2025181155A1 (en) 2024-02-26 2025-09-04 Vib Vzw Human beta-glucocerebrosidase binders and uses thereof
AU2025229047A1 (en) 2024-02-29 2026-03-05 Octapharma Ag Cystine knot domain fusion protein dimers
WO2025186787A1 (en) 2024-03-08 2025-09-12 Momenta Pharmaceuticals, Inc. Compositions and methods for treating fetal and neonatal alloimmune thrombocytopenia
GB202403780D0 (en) 2024-03-15 2024-05-01 Avacta Life Sciences Ltd Polypeptides and methods for detecting and quantifying small molecules
WO2025196308A1 (en) 2024-03-22 2025-09-25 Vib Vzw Means and methods for displaying fc-containing proteins on cells and selection thereof
WO2025207946A1 (en) 2024-03-28 2025-10-02 Genzyme Corporation Polypeptides binding to a specific epitope of the transferrin receptor 1
TW202602941A (en) 2024-04-10 2026-01-16 美商奧迪希治療公司 Anti-tnfr2 antigen-binding proteins and uses thereof
WO2025217497A1 (en) 2024-04-12 2025-10-16 Paragon Therapeutics, Inc. Albumin binding proteins and methods of use
WO2025219231A1 (en) 2024-04-15 2025-10-23 Vib Vzw Computer-implemented means and methods for the de novo design of antibodies targeting a specific epitope
WO2025229160A1 (en) 2024-05-01 2025-11-06 Commit Biologics Aps Engineered proteins that engage complement factor and a protein antigen, methods, and uses thereof
WO2025255558A2 (en) 2024-06-07 2025-12-11 Odyssey Therapeutics, Inc. Anti-thymic stromal lymphopoietin (tslp) antigen-binding proteins and uses thereof
WO2025255435A2 (en) 2024-06-07 2025-12-11 Odyssey Therapeutics, Inc. Antigen-binding proteins against serum albumin and uses thereof
WO2026006708A2 (en) 2024-06-27 2026-01-02 Odyssey Therapeutics, Inc. Anti-cd25 antigen-binding proteins and uses thereof
WO2026006809A1 (en) 2024-06-27 2026-01-02 Odyssey Therapeutics, Inc. Multispecific molecules binding tnfr2 and cd25 and uses thereof
WO2026008665A1 (en) 2024-07-01 2026-01-08 Vib Vzw Binders of the pd-1•pd-l1 complex and their use
WO2026008785A1 (en) 2024-07-03 2026-01-08 Biotalys NV Agrochemical compositions
WO2026017728A1 (en) 2024-07-16 2026-01-22 Synapse Research Institute Polypeptide for treatment of microvascular thrombosis
WO2026030375A2 (en) 2024-07-30 2026-02-05 Genzyme Corporation Lipid nanoparticles and methods of manufacture and use thereof
WO2026027659A1 (en) 2024-07-31 2026-02-05 Seni-Preps B.V. Improved immunoglobulin single variable domains that inhibit urease and use thereof
WO2026052756A1 (en) 2024-09-05 2026-03-12 Ablynx Nv Her2 binding immunoglobulin single variable domains, constructs comprising the same and uses thereof
WO2026068859A1 (en) 2024-09-30 2026-04-02 Université Libre de Bruxelles Wnt signaling agonist molecules in the treatment of a bone-related disease or disorder
WO2026083290A1 (en) 2024-10-16 2026-04-23 Momenta Pharmaceuticals, Inc. Compositions and methods for treating sjögren's disease
WO2026087355A1 (en) 2024-10-22 2026-04-30 Biotalys NV Downstream processing

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4405306A (en) * 1981-12-08 1983-09-20 Beecham Inc. Medicated disposable douche product
US5091513A (en) * 1987-05-21 1992-02-25 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5196193A (en) * 1989-10-31 1993-03-23 Ophidian Pharmaceuticals, Inc. Antivenoms and methods for making antivenoms
US5487890A (en) * 1990-10-04 1996-01-30 University Of Virginia Patent Foundation Mammalian primate erythrocyte bound heteropolymerized monoclonal antibodies and methods of use thereof
US5656273A (en) * 1994-01-18 1997-08-12 Genentech, Inc. Method of treatment of parasitic infection using IgE antagonists
US5672347A (en) * 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use
US5837243A (en) * 1995-06-07 1998-11-17 Medarex, Inc. Therapeutic compounds comprised of anti-Fc receptor antibodies
US5843440A (en) * 1990-10-03 1998-12-01 Redcell Canada, Inc. Cellular and serum protein anchors for modulating pharmacokinetics
US5891996A (en) * 1972-09-17 1999-04-06 Centro De Inmunologia Molecular Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (EGF-R); diagnostic and therapeutic use
US5976532A (en) * 1988-05-18 1999-11-02 Centocor, Inc. Method of antithrombotic therapy using anti-GPIIb/IIIa antibodies or fragments thereof, including c7E3
US5994511A (en) * 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
US6066718A (en) * 1992-09-25 2000-05-23 Novartis Corporation Reshaped monoclonal antibodies against an immunoglobulin isotype
US6251393B1 (en) * 1998-10-23 2001-06-26 The Brigham And Women's Hospital, Inc. Conformation-specific anti-von Willebrand Factor antibodies
US20020001587A1 (en) * 2000-03-16 2002-01-03 Sharon Erickson Methods of treatment using anti-ErbB antibody-maytansinoid conjugates
US20020009453A1 (en) * 2000-06-16 2002-01-24 Symphogen A/S Polyclonal antibody composition for treating allergy
US20020028204A1 (en) * 1994-11-30 2002-03-07 Ajinomoto Co., Inc. Antithrombotic agent and anti-von willebrand factor monoclonal antibody
US20020058033A1 (en) * 2000-10-13 2002-05-16 Raisch Kevin Paul Human anti-epidermal growth factor receptor single-chain antibodies
US20020076404A1 (en) * 1998-01-29 2002-06-20 Chang Tse Wen Treating atopic dermatitis with IgE antagonists
US6419934B1 (en) * 1999-02-24 2002-07-16 Edward L. Tobinick TNF modulators for treating neurological disorders associated with viral infection
US20020132275A1 (en) * 2000-11-03 2002-09-19 Board Of Regents, The University Of Texas System Methods for detecting the efficacy of anticancer treatments
US20020165387A1 (en) * 1996-10-31 2002-11-07 Kerr Anderson W. H. High affinity humanized anti-CEA monoclonal antibodies
US6504013B1 (en) * 2000-02-01 2003-01-07 Idexx Laboratories, Inc. Canine allergy therapeutic recombinant chimeric anti-IgE monoclonal antibody
US20030092892A1 (en) * 1997-10-27 2003-05-15 Frenken Leon Gerardus Multivalent antigen-binding proteins
US6759518B1 (en) * 1998-04-09 2004-07-06 Vectron Therapeutics Ag Single-chain multiple antigen-binding molecule, its preparation and use
US20040180046A1 (en) * 2000-04-26 2004-09-16 Jeff Himawan Bispecific molecules and uses thereof
US20040197326A1 (en) * 1995-07-27 2004-10-07 Genentech, Inc. Method for treatment of allergic asthma
US20040219643A1 (en) * 2001-06-28 2004-11-04 Greg Winter Dual-specific ligand
US6902734B2 (en) * 2000-08-07 2005-06-07 Centocor, Inc. Anti-IL-12 antibodies and compositions thereof
US20060034833A1 (en) * 2002-11-08 2006-02-16 Els Beirnaert Single domain antibodies directed against interferron-gamma and uses therefor
US20060034845A1 (en) * 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
US20060115470A1 (en) * 2002-11-08 2006-06-01 Ablynx N. V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
US7084257B2 (en) * 2001-10-05 2006-08-01 Amgen Inc. Fully human antibody Fab fragments with human interferon-gamma neutralizing activity
US20060228355A1 (en) * 2003-11-07 2006-10-12 Toon Laeremans Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
US20070264253A1 (en) * 2004-03-19 2007-11-15 Meilin Liu Human Anti-Epidermal Growth Factor Receptor Antibody
US7300655B2 (en) * 2002-08-01 2007-11-27 Immunomedics, Inc. Alpha-fetoprotein Immu31 antibodies and fusion proteins and methods of use thereof
US7368111B2 (en) * 1995-10-06 2008-05-06 Cambridge Antibody Technology Limited Human antibodies specific for TGFβ2
US7589180B2 (en) * 2001-05-11 2009-09-15 Abbott Laboratories Inc. Specific binding proteins and uses thereof
US20090238829A1 (en) * 2002-11-08 2009-09-24 Ablynx N.V. Stabilized single domain antibodies
US20100003253A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US7897151B2 (en) * 2000-08-30 2011-03-01 Pharmacia & Upjohn Company, Llc Anti-IgE vaccines

Family Cites Families (162)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US460167A (en) 1891-09-29 Car-mover
SE307996B (en) 1965-11-30 1969-01-27 Asea Ab
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
CA1204148A (en) 1982-05-26 1986-05-06 Petrus R. Nelen Apparatus for transferring xerographic images
US4559157A (en) 1983-04-21 1985-12-17 Creative Products Resource Associates, Ltd. Cosmetic applicator useful for skin moisturizing
LU84979A1 (en) 1983-08-30 1985-04-24 Oreal COSMETIC OR PHARMACEUTICAL COMPOSITION IN AQUEOUS OR ANHYDROUS FORM WHOSE FATTY PHASE CONTAINS OLIGOMER POLYETHER AND NEW OLIGOMER POLYETHERS
GB2148299B (en) 1983-09-01 1988-01-06 Hybritech Inc Antibody compositions of therapeutic agents having an extended serum half-life
US4946788A (en) 1985-06-11 1990-08-07 Ciba-Geigy Corporation Purified immunoglobulin-related factor, novel monoclonal antibodies, hybridoma cell lines, processes and applications
US4714759A (en) 1985-12-02 1987-12-22 Whitaker Jr Robert B Immunotoxin therapy of allergy
JPS62175426A (en) 1986-01-27 1987-08-01 Kazufumi Shimizu Antibody and spraying agent containing said substance as active component
US5238919A (en) 1986-05-30 1993-08-24 Scipps Clinic And Research Foundation Peptides that inhibit von Willebrand Factor binding to the platelet SPIB receptor
JP2647427B2 (en) 1987-04-24 1997-08-27 帝人株式会社 Detection method, monoclonal antibody, and detection kit for determining disease state of subject
US4940782A (en) 1987-06-08 1990-07-10 G. D. Searle & Co. Monoclonal antibodies against IgE-associated determinants, hybrid cell lines producing these antibodies, and use therefore
DE3853740T2 (en) 1987-06-10 1995-11-09 Dana Farber Cancer Inst Inc Bifunctional antibody designs and methods for the selective killing of cell populations.
US5200510A (en) 1987-06-16 1993-04-06 Zymogenetics, Inc. Method for purifying factor viii:c, von willebrand factor and complexes thereof
US4820508A (en) 1987-06-23 1989-04-11 Neutrogena Corporation Skin protective composition
EP0300191A1 (en) 1987-07-17 1989-01-25 Siemens Aktiengesellschaft Device for storing, isolating and feeding components loadable in bulk
GB8725529D0 (en) 1987-10-30 1987-12-02 Delta Biotechnology Ltd Polypeptides
US4962035A (en) 1987-12-01 1990-10-09 President And Fellows Of Harvard College DNA encoding IgE receptor alpha-subunit or fragment thereof
US5428133A (en) 1987-12-31 1995-06-27 Tanox Biosystems, Inc. Chimeric anti-human IgE-monoclonal antibody which binds to secreted IgE and membrane-bound IgE expressed by IgE-expressing B cells but notto IgE bound to FC receptors on basophils
DE122006000007I2 (en) 1987-12-31 2008-03-27 Tanox Inc BEARING B-LYMPHOCYTES.
US5252467A (en) 1987-12-31 1993-10-12 Tanox Biosystems, Inc. Method of making antibodies to antigenic epitopes of IGE present on B cells but not basophil cell surface or secreted, soluble IGE
US5091313A (en) 1988-08-05 1992-02-25 Tanox Biosystems, Inc. Antigenic epitopes of IgE present on B cell but not basophil surface
US5231026A (en) 1987-12-31 1993-07-27 Tanox Biosystems, Inc. DNA encoding murine-human chimeric antibodies specific for antigenic epitopes of IgE present on the extracellular segment of the membrane domain of membrane-bound IgE
US5422258A (en) 1987-12-31 1995-06-06 Tanox Biosystems, Inc. Methods for producing high affinity anti-human IgE-monoclonal antibodies which binds to IgE on IgEabearing B cells but not basophils
US4992478A (en) 1988-04-04 1991-02-12 Warner-Lambert Company Antiinflammatory skin moisturizing composition and method of preparing same
JPH01268645A (en) 1988-04-18 1989-10-26 Teijin Ltd Agent for suppressing schwartzman reaction
US4938949A (en) 1988-09-12 1990-07-03 University Of New York Treatment of damaged bone marrow and dosage units therefor
EP0366043B1 (en) 1988-10-24 1994-03-30 Otsuka Pharmaceutical Co., Ltd. Monoclonal antibody
US5051264A (en) * 1988-11-02 1991-09-24 Collaborative Research, Inc. Potentiation of the thrombolytic effect of prourokinase type plasminogen activators by streptokinase
ES2052027T5 (en) 1988-11-11 2005-04-16 Medical Research Council IMMUNOGLOBULINE VARIABLE DOMAIN SEQUENCE CLONING.
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
GB8905400D0 (en) * 1989-03-09 1989-04-19 Jonker Margreet Medicaments
US5766883A (en) 1989-04-29 1998-06-16 Delta Biotechnology Limited Polypeptides
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5032666A (en) * 1989-06-19 1991-07-16 Becton, Dickinson And Company Amine rich fluorinated polyurethaneureas and their use in a method to immobilize an antithrombogenic agent on a device surface
SE509359C2 (en) 1989-08-01 1999-01-18 Cemu Bioteknik Ab Use of stabilized protein or peptide conjugates for the preparation of a drug
US6267964B1 (en) 1989-08-01 2001-07-31 Affibody Technology Sweden Ab Stabilized protein or peptide conjugates able to bond albumin having extended biological half-lives
US6498237B2 (en) 1989-08-07 2002-12-24 Peptech Limited Tumor necrosis factor antibodies
US5644034A (en) * 1989-08-07 1997-07-01 Peptide Technology Ltd. Tumour necrosis factor binding ligands
JP3443119B2 (en) * 1989-08-07 2003-09-02 ペプテック リミテッド Tumor necrosis factor binding ligand
GB8921123D0 (en) * 1989-09-19 1989-11-08 Millar Ann B Treatment of ards
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
GB8928874D0 (en) 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
GB9012995D0 (en) 1990-06-11 1990-08-01 Celltech Ltd Multivalent antigen-binding proteins
GB9016299D0 (en) 1990-07-25 1990-09-12 Brien Caroline J O Binding substances
JP2988635B2 (en) 1990-09-18 1999-12-13 塩野義製薬株式会社 Monoclonal antibody against human IgE
US5612034A (en) 1990-10-03 1997-03-18 Redcell, Inc. Super-globuling for in vivo extended lifetimes
US5656272A (en) 1991-03-18 1997-08-12 New York University Medical Center Methods of treating TNF-α-mediated Crohn's disease using chimeric anti-TNF antibodies
DE69230998T2 (en) 1991-03-21 2000-12-21 Masimo Corp., Laguna Hills LOW-NOISE OPTICAL CONVERTER
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
ES2109362T3 (en) 1991-06-21 1998-01-16 Univ Cincinnati ADMINISTRABLE PROTEINS ORALLY AND METHOD TO MAKE THEM.
DK0602126T3 (en) 1991-08-14 2003-06-16 Genentech Inc Immunoglobulin variants for specific FC epsilon receptors
DE69232859T2 (en) * 1991-09-13 2003-04-10 Chiron Corp. (N.D.Ges.D. Staates Delaware), Emeryville COMPOSITION OF SEVERAL IMMUNE REACTIVE HEPATITIS C VIRUS POLYPEPTIDES
DE4293170T1 (en) * 1991-10-01 1993-11-18 Otsuka Pharma Co Ltd Antithrombotic resin, antithrombotic tube, antithrombotic film and antithrombotic coating
DE69233528T2 (en) 1991-11-25 2006-03-16 Enzon, Inc. Process for the preparation of multivalent antigen-binding proteins
JP4157160B2 (en) 1991-12-13 2008-09-24 ゾーマ テクノロジー リミテッド Methods for the preparation of modified antibody variable regions
US5869619A (en) 1991-12-13 1999-02-09 Xoma Corporation Modified antibody variable domains
EP0584421A1 (en) 1992-08-21 1994-03-02 Cécile Casterman Immunoglobulins devoid of light chains
PT1621554E (en) 1992-08-21 2009-07-13 Univ Bruxelles IMMUNOGLOBTAINES OF LIGHT CHAINS
US6765087B1 (en) * 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
ES2121907T3 (en) 1992-08-28 1998-12-16 Bayer Ag USE OF ANTI-TNF MONOCLONAL ANTIBODIES FOR THE TREATMENT OF BACTERIAL MENINGITIS.
PT589840E (en) 1992-09-24 2004-08-31 Tanox Biosystems Inc MONOCLONAL ANTIBODIES REFORMULATED AGAINST AN IMMUNOGLOBULIN ISOTIPE
WO1994008619A1 (en) 1992-10-08 1994-04-28 The Kennedy Institute Of Rheumatology Treatment of autoimmune and inflammatory disorders
GB9221657D0 (en) 1992-10-15 1992-11-25 Scotgen Ltd Recombinant bispecific antibodies
AU5669694A (en) * 1992-11-20 1994-06-22 Schering Corporation Antagonists of human gamma interferon
JP3720353B2 (en) 1992-12-04 2005-11-24 メディカル リサーチ カウンシル Multivalent and multispecific binding proteins, their production and use
DE69327229T2 (en) 1992-12-11 2000-03-30 The Dow Chemical Co., Midland Multivalent single chain antibodies
DE69322289T2 (en) 1992-12-29 1999-05-20 Genentech, Inc., South San Francisco, Calif. TREATMENT OF INFLAMMABLE INTESTINAL DISEASES WITH INTERFERON GAMMA INHIBITORS
MX9401351A (en) 1993-02-22 1994-08-31 Alza Corp COMPOSITIONS FOR ORAL SUPPLY FOR ACTIVE AGENTS.
US5888511A (en) 1993-02-26 1999-03-30 Advanced Biotherapy Concepts, Inc. Treatment of autoimmune diseases, including AIDS
EP0614984B2 (en) 1993-03-05 2010-11-03 Bayer HealthCare LLC Anti-TNF alpha human monoclonal antibodies
US6838254B1 (en) * 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
EP0698097B1 (en) 1993-04-29 2001-08-16 Unilever N.V. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
GB9311454D0 (en) 1993-06-03 1993-07-21 Agricultural & Food Res Pharmaceutical compositions
DK104093D0 (en) * 1993-09-17 1993-09-17 Osteometer A S PROCEDURE FOR DETERMINING COLLAGEN FRAGMENTS IN BODY LIQUIDS, TEST KITS AND MEANS FOR EXERCISING THE PROCEDURE AND USING THE PROCEDURE FOR DIAGNOSTICING THE DISEASES OF TABLET METAL
WO1995017673A1 (en) 1993-12-21 1995-06-29 Ocutech, Inc. Ocular diagnostics and therapies
US5747148A (en) 1994-09-12 1998-05-05 Minnesota Mining And Manufacturing Company Ink jet printing sheet
US5670132A (en) * 1994-09-20 1997-09-23 Immunomedics, Inc. Modified radioantibody fragments for reduced renal uptake
US6544513B2 (en) * 1994-09-26 2003-04-08 The State Of New South Wales C/- Nsw Department Of Agriculture Method of controlling moth and other insect pests
US5877155A (en) * 1995-03-17 1999-03-02 The Research Foundation Of State University Of New York Mimotopes and anti-mimotopes of human platelet glycoprotein Ib/IX
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
US6165463A (en) 1997-10-16 2000-12-26 Inhale Therapeutic Systems, Inc. Dispersible antibody compositions and methods for their preparation and use
EP0739981A1 (en) 1995-04-25 1996-10-30 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
JPH11507535A (en) * 1995-06-07 1999-07-06 イムクローン システムズ インコーポレイテッド Antibodies and antibody fragments that suppress tumor growth
US5637038A (en) 1995-07-10 1997-06-10 Davis; James F. Apparatus and method for skinning poultry
ZA966075B (en) 1995-07-27 1998-01-19 Genentech Inc Protein formulation.
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
GB9518323D0 (en) 1995-09-07 1995-11-08 Steidler Lothar Materials and methods relating to the attachment and display of substances on cell surfaces
ATE194291T1 (en) * 1995-09-18 2000-07-15 Intracel Corp NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST RESPIRATORY SYNZYTIAL VIRUS
HU230048B1 (en) 1996-02-09 2015-06-29 Abbvie Biotechnology Ltd Use of human tnf-alpha binding antibodies
WO1997038102A1 (en) 1996-04-04 1997-10-16 Unilever Plc Multivalent and multispecific antigen-binding protein
JP2000515002A (en) 1996-06-27 2000-11-14 フラームス・インテルウニフェルジテール・インスティテュート・フォール・ビオテヒノロジー・ヴェーゼットウェー Recognition molecules that specifically interact with the active site or cleft of the target molecule
US6361938B1 (en) 1996-11-08 2002-03-26 Elan Corporation, Plc Peptides which enhance transport across tissues and methods of identifying and using the same
IT1289608B1 (en) 1997-02-05 1998-10-15 Angelini Ricerche Spa COMPOSITION FOR THERAPEUTIC OR DIAGNOSTIC USE ADMINISTRABLE VIA INTRANASAL, SUBLINGUAL OR VAGINAL
US5942602A (en) * 1997-02-13 1999-08-24 Schering Aktiengessellschaft Growth factor receptor antibodies
AUPO561697A0 (en) 1997-03-13 1997-04-10 Crc For Cardiac Technology Haemocompatible surfaces
BR9809932A (en) 1997-06-05 2000-08-01 Lilly Co Eli Methods for treating thrombotic disorders
DE29712318U1 (en) 1997-07-07 1997-10-02 Arnold, Günter, 24977 Langballig Paring knife
WO1999009055A2 (en) * 1997-08-18 1999-02-25 Innogenetics N.V. Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders
ATE535154T1 (en) 1998-03-12 2011-12-15 Vhsquared Ltd PRODUCTS THAT CONTAIN INACTIVATED YEAST OR MOLD THAT HAVE ACTIVE ANTIBODIES ON THEIR EXTERNAL SURFACE
EP0954978B1 (en) 1998-03-12 2011-11-30 VHsquared Limited New products comprising inactivated yeasts or moulds provided with active antibodies
CA2328725A1 (en) 1998-04-15 1999-10-21 Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
WO1999064069A1 (en) * 1998-06-10 1999-12-16 Ophidian Pharmaceuticals, Inc. Antibodies to cytokines in the prevention and treatment of inflammatory bowel disease
DE69905291T3 (en) 1998-07-06 2009-06-25 Perkinelmer Cellular Sciences Belgium Sprl Bioluminescence assay for agonists or antagonists of a calcium-compound receptor
US6228360B1 (en) 1998-08-19 2001-05-08 Ajinomoto Co., Inc. Antithrombotic agent and humanized anti-von Willebrand factor monoclonal antibody
US6149934A (en) * 1999-04-23 2000-11-21 Kimberly-Clark Worldwide, Inc. Absorbent article having a lotionized bodyside liner
ATE259829T1 (en) 1998-10-20 2004-03-15 Vlaams Interuniv Inst Biotech USE OF A CYTOKINE-PRODUCING LACTOCOCCUS STRAIN FOR THE TREATMENT OF COLITIS
EP1002861A1 (en) 1998-10-26 2000-05-24 Unilever Plc Antigen-binding proteins comprising a linker which confers restricted conformational flexibility
GB9824632D0 (en) 1998-11-10 1999-01-06 Celltech Therapeutics Ltd Biological compounds
IL127127A0 (en) 1998-11-18 1999-09-22 Peptor Ltd Small functional units of antibody heavy chain variable regions
DE19852800C1 (en) 1998-11-16 2000-04-13 Univ Albert Ludwigs Freiburg Production of antibodies to a polypeptide encoded by a known DNA sequence comprises binding of antibodies produced by DNA vaccination to immobilized recombinantly expressed polypeptide
EP1135415B1 (en) 1998-12-01 2010-08-11 Facet Biotech Corporation Humanized antibodies to gamma-interferon
ATE330631T1 (en) 1999-01-05 2006-07-15 Univ Southern Australia ANTIBODIES FRAGMENTS FOR THE LOCAL TREATMENT OF EYE DISEASES
CN1252264C (en) 1999-04-22 2006-04-19 荷兰联合利华有限公司 Inhibition of viral infection using monovalent antigen-binding proteins
US7622259B1 (en) 1999-07-05 2009-11-24 K.U. Leuven Research & Development Detection of von-Willebrand factor (vWF) activity
GB9918788D0 (en) 1999-08-10 1999-10-13 Leuven K U Res & Dev Antithrombotic effect of platelet glycoprotein 1b blocking monoclonal Fab fragments
US6436401B1 (en) * 1999-09-14 2002-08-20 Milkhaus Laboratory, Inc. Methods for alleviating symptoms associated with diabetes and diabetic neuropathy comprising administration of low levels of antibodies
EP1212100B1 (en) 1999-09-16 2005-04-06 Unilever Plc Delivery system for antidandruff agent
EP1118669A3 (en) 1999-12-17 2001-08-29 Unilever Plc Production of camelid antibodies in plants
US7608681B2 (en) 1999-12-24 2009-10-27 Genentech, Inc. Methods and compositions for prolonging elimination half-times of bioactive compounds
PL356836A1 (en) 2000-02-10 2004-07-12 Abbott Laboratories Antibodies that bind human interleukin-18 and methods of making and using
EP1134231B1 (en) 2000-03-14 2009-04-15 Unilever N.V. Antibody heavy chain variable domains against human dietary lipases, and their uses
JP2001318093A (en) * 2000-05-09 2001-11-16 Keio Gijuku Anti-inflammatory agent containing a platelet GPIbα function inhibitor as an active ingredient
AU2001264747A1 (en) 2000-05-22 2001-12-03 Idec Pharmaceuticals Corporation Identification of unique binding interactions between certain antibodies and thehuman b7.1 and b7.2 co-stimulatory antigens
US7943129B2 (en) * 2000-05-26 2011-05-17 National Research Council Of Canada Single-domain brain-targeting antibody fragments derived from llama antibodies
UA81743C2 (en) 2000-08-07 2008-02-11 Центокор, Инк. HUMAN MONOCLONAL ANTIBODY WHICH SPECIFICALLY BINDS TUMOR NECROSIS FACTOR ALFA (TNFα), PHARMACEUTICAL MIXTURE CONTAINING THEREOF, AND METHOD FOR TREATING ARTHRITIS
MY128992A (en) 2000-08-25 2007-03-30 Merck Patent Gmbh Saratin for inhibiting platelet adhesion to collagen
JP2004508036A (en) 2000-09-08 2004-03-18 マイクロメット　アーゲー Antibodies and / or chemokine constructs and their use in immune disorders
US6545097B2 (en) * 2000-12-12 2003-04-08 Scimed Life Systems, Inc. Drug delivery compositions and medical devices containing block copolymer
ATE513854T1 (en) 2000-12-13 2011-07-15 Bac Ip B V PROTEIN GRID OF VARIABLE DOMAIN OF THE CAMEL IMMUNOGLOBULIN HEAVY CHAIN
GB0031448D0 (en) * 2000-12-22 2001-02-07 Leuven K U Res & Dev Inhibition of the vWF-collagen interaction by anti-human vWF monoclonal antibody (82D6A3) results in abolition of in vivo arterial platelet thrombus formation
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US6632470B2 (en) * 2001-01-31 2003-10-14 Percardia Methods for surface modification
CA2442074C (en) * 2001-03-28 2014-07-22 Heska Corporation Methods of detecting early renal disease in animals
US20050019324A1 (en) * 2001-03-29 2005-01-27 Wreschner Daniel H. Peptides and antibodies to muc 1 proteins
US8178098B2 (en) * 2001-04-03 2012-05-15 National Jewish Health Method to inhibit airway hyperresponsiveness using aerosolized T cell receptor antibodies
WO2002081649A2 (en) * 2001-04-06 2002-10-17 The Trustees Of The University Of Pennsylvania ErbB INTERFACE PEPTIDOMIMETICS AND METHODS OF USE THEREOF
JP2005289809A (en) * 2001-10-24 2005-10-20 Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) Mutant heavy chain antibody
JP4323317B2 (en) 2001-12-21 2009-09-02 フラームス・インテルウニフェルシタイル・インステイチュート・フォール・ビオテヒノロヒー・ヴェーゼットウェー（ヴェーイーベー・ヴェーゼットウェー） Methods for cloning variable region sequences
US20040063912A1 (en) 2002-03-15 2004-04-01 The Brigham And Women's Hospital, Inc. Central airway administration for systemic delivery of therapeutics
WO2003077834A2 (en) 2002-03-15 2003-09-25 The Brigham And Women's Hospital, Inc. Central airway administration for systemic delivery of therapeutics
EP1600459A3 (en) 2002-06-28 2005-12-07 Domantis Limited Ligand
US20050136056A1 (en) 2002-07-29 2005-06-23 Shunsuke Kageyama Pharmaceutical composition for the treatment of thrombocytopenia
AU2003251238A1 (en) 2002-08-07 2004-02-25 Umc Utrecht Holding B.V. Modulation of platelet adhesion based on the surface exposed beta-switch loop of platelet glycoprotein ib-alpha
US9320792B2 (en) * 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
SI1639011T1 (en) 2003-06-30 2009-04-30 Domantis Ltd Pegylated single domain antibodies (dAb)
US7566701B2 (en) 2004-09-07 2009-07-28 Archemix Corp. Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics
AP2007004019A0 (en) 2004-11-25 2007-06-30 Unilever Plc Heavy chain and single domain antibodies
CA2588892A1 (en) 2004-12-02 2006-06-08 Dormantis Limited Anti-il-1r1 single domain antibodies and therapeutic uses
CA2593328C (en) 2005-01-14 2018-02-06 Ablynx N.V. Methods and assays for distinguishing between different forms of diseases and disorders characterized by thrombocytopenia and/or by spontaneous interaction between von willebrand factor (vwf) and platelets
AU2006249144B2 (en) 2005-05-18 2011-11-17 Ablynx Nv Improved NanobodiesTM against Tumor Necrosis Factor-alpha
MX2007014564A (en) 2005-05-20 2008-02-07 Ablynx Nv VHH ANTIBODIES OF SINGLE DOMAIN AGAINST VON WILLEBRAND FACTOR.
DE102005023617A1 (en) 2005-05-21 2006-11-23 Aspre Ag Method for mixing colors in a display
EP1754955B1 (en) 2005-08-19 2015-05-06 Aisin Aw Co., Ltd. Navigation method and corresponding system for determining a travel related time
CA2632424A1 (en) 2005-12-06 2007-06-14 Domantis Limited Bispecific ligands with binding specificity to cell surface targets and methods of use therefor
WO2008049881A2 (en) 2006-10-25 2008-05-02 Umc Utrecht Holding Bv Polypeptides and pharmaceutical compositions comprising the same for the prevention and treatment of complications associated with infectious diseases
TW200938222A (en) 2007-12-13 2009-09-16 Glaxo Group Ltd Compositions for pulmonary delivery
CA2718081A1 (en) 2008-03-21 2009-09-24 Ablynx Nv Von willebrand factor specific binders and methods of use therefor
CN102089325A (en) 2008-04-17 2011-06-08 埃博灵克斯股份有限公司 Peptides capable of binding to serum proteins and compounds, constructs and polypeptides comprising the same
US20120321640A1 (en) 2009-12-01 2012-12-20 Ablynx N.V. Von willebrand factor specific binding agents and uses thereof

Patent Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891996A (en) * 1972-09-17 1999-04-06 Centro De Inmunologia Molecular Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (EGF-R); diagnostic and therapeutic use
US4405306A (en) * 1981-12-08 1983-09-20 Beecham Inc. Medicated disposable douche product
US5672347A (en) * 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use
US5091513A (en) * 1987-05-21 1992-02-25 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5976532A (en) * 1988-05-18 1999-11-02 Centocor, Inc. Method of antithrombotic therapy using anti-GPIIb/IIIa antibodies or fragments thereof, including c7E3
US5196193A (en) * 1989-10-31 1993-03-23 Ophidian Pharmaceuticals, Inc. Antivenoms and methods for making antivenoms
US5843440A (en) * 1990-10-03 1998-12-01 Redcell Canada, Inc. Cellular and serum protein anchors for modulating pharmacokinetics
US5487890A (en) * 1990-10-04 1996-01-30 University Of Virginia Patent Foundation Mammalian primate erythrocyte bound heteropolymerized monoclonal antibodies and methods of use thereof
US6066718A (en) * 1992-09-25 2000-05-23 Novartis Corporation Reshaped monoclonal antibodies against an immunoglobulin isotype
US5656273A (en) * 1994-01-18 1997-08-12 Genentech, Inc. Method of treatment of parasitic infection using IgE antagonists
US20020028204A1 (en) * 1994-11-30 2002-03-07 Ajinomoto Co., Inc. Antithrombotic agent and anti-von willebrand factor monoclonal antibody
US5837243A (en) * 1995-06-07 1998-11-17 Medarex, Inc. Therapeutic compounds comprised of anti-Fc receptor antibodies
US20040197326A1 (en) * 1995-07-27 2004-10-07 Genentech, Inc. Method for treatment of allergic asthma
US7368111B2 (en) * 1995-10-06 2008-05-06 Cambridge Antibody Technology Limited Human antibodies specific for TGFβ2
US20020165387A1 (en) * 1996-10-31 2002-11-07 Kerr Anderson W. H. High affinity humanized anti-CEA monoclonal antibodies
US5994511A (en) * 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
US20020054878A1 (en) * 1997-07-02 2002-05-09 Genentech, Inc. Anti-IgE antibodies
US6670453B2 (en) * 1997-10-27 2003-12-30 Unilever Patent Holdings B.V. Multivalent antigen-binding proteins
US20030092892A1 (en) * 1997-10-27 2003-05-15 Frenken Leon Gerardus Multivalent antigen-binding proteins
US20020076404A1 (en) * 1998-01-29 2002-06-20 Chang Tse Wen Treating atopic dermatitis with IgE antagonists
US6759518B1 (en) * 1998-04-09 2004-07-06 Vectron Therapeutics Ag Single-chain multiple antigen-binding molecule, its preparation and use
US20010024647A1 (en) * 1998-10-23 2001-09-27 Handin Robert I. Conformation-specific anti-von Willebrand factor antibodies
US6251393B1 (en) * 1998-10-23 2001-06-26 The Brigham And Women's Hospital, Inc. Conformation-specific anti-von Willebrand Factor antibodies
US6419934B1 (en) * 1999-02-24 2002-07-16 Edward L. Tobinick TNF modulators for treating neurological disorders associated with viral infection
US6504013B1 (en) * 2000-02-01 2003-01-07 Idexx Laboratories, Inc. Canine allergy therapeutic recombinant chimeric anti-IgE monoclonal antibody
US20020001587A1 (en) * 2000-03-16 2002-01-03 Sharon Erickson Methods of treatment using anti-ErbB antibody-maytansinoid conjugates
US20040180046A1 (en) * 2000-04-26 2004-09-16 Jeff Himawan Bispecific molecules and uses thereof
US20020009453A1 (en) * 2000-06-16 2002-01-24 Symphogen A/S Polyclonal antibody composition for treating allergy
US6902734B2 (en) * 2000-08-07 2005-06-07 Centocor, Inc. Anti-IL-12 antibodies and compositions thereof
US7897151B2 (en) * 2000-08-30 2011-03-01 Pharmacia & Upjohn Company, Llc Anti-IgE vaccines
US20020058033A1 (en) * 2000-10-13 2002-05-16 Raisch Kevin Paul Human anti-epidermal growth factor receptor single-chain antibodies
US20020132275A1 (en) * 2000-11-03 2002-09-19 Board Of Regents, The University Of Texas System Methods for detecting the efficacy of anticancer treatments
US7589180B2 (en) * 2001-05-11 2009-09-15 Abbott Laboratories Inc. Specific binding proteins and uses thereof
US20040219643A1 (en) * 2001-06-28 2004-11-04 Greg Winter Dual-specific ligand
US7084257B2 (en) * 2001-10-05 2006-08-01 Amgen Inc. Fully human antibody Fab fragments with human interferon-gamma neutralizing activity
US7300655B2 (en) * 2002-08-01 2007-11-27 Immunomedics, Inc. Alpha-fetoprotein Immu31 antibodies and fusion proteins and methods of use thereof
US20090022721A1 (en) * 2002-11-08 2009-01-22 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
US20100003248A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V Polypeptide constructs for rectal and/or vaginal administration
US20110184150A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US20070077249A1 (en) * 2002-11-08 2007-04-05 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
US20110184145A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Method of administering therapeutic polypeptides, and polypeptides therefor
US20060115470A1 (en) * 2002-11-08 2006-06-01 Ablynx N. V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
US20060034845A1 (en) * 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
US20090238829A1 (en) * 2002-11-08 2009-09-24 Ablynx N.V. Stabilized single domain antibodies
US20110184151A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20090324512A1 (en) * 2002-11-08 2009-12-31 Ablynx N.V. Polypeptide constructs for nasal administration
US20100003253A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20070237769A1 (en) * 2002-11-08 2007-10-11 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
US20100003249A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Polypeptide constructs for topical administration
US20100021459A1 (en) * 2002-11-08 2010-01-28 Ablynx N.V. Polypeptide constructs for intracellular delivery
US20100040613A1 (en) * 2002-11-08 2010-02-18 Ablynx N.V. Polypeptide constructs for sublingual administration
US20110027281A1 (en) * 2002-11-08 2011-02-03 Ablynx N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US20060034833A1 (en) * 2002-11-08 2006-02-16 Els Beirnaert Single domain antibodies directed against interferron-gamma and uses therefor
US20110123529A1 (en) * 2002-11-08 2011-05-26 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20110178277A1 (en) * 2002-11-08 2011-07-21 Ablynx N.V. Stabilized single domain antibodies
US20060228355A1 (en) * 2003-11-07 2006-10-12 Toon Laeremans Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
US7598350B2 (en) * 2004-03-19 2009-10-06 Imclone Llc Human anti-epidermal growth factor receptor antibody
US20070264253A1 (en) * 2004-03-19 2007-11-15 Meilin Liu Human Anti-Epidermal Growth Factor Receptor Antibody

Cited By (188)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9156905B2 (en) 2001-10-24 2015-10-13 Vib Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
US8097251B2 (en) 2001-10-24 2012-01-17 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
US20070031424A1 (en) * 2001-10-24 2007-02-08 Vlaams Interuniversitair Instituut Voor Biotechnogie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
US20050054001A1 (en) * 2001-10-24 2005-03-10 Serge Muyldermans Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
US10041102B2 (en) 2002-10-08 2018-08-07 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
US20090238829A1 (en) * 2002-11-08 2009-09-24 Ablynx N.V. Stabilized single domain antibodies
US20110184145A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Method of administering therapeutic polypeptides, and polypeptides therefor
US9243065B2 (en) 2002-11-08 2016-01-26 Ablynx N.V. Polypeptide constructs including VHH directed against EGFR for intracellular delivery
US20090022721A1 (en) * 2002-11-08 2009-01-22 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
US9320792B2 (en) 2002-11-08 2016-04-26 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US20090324512A1 (en) * 2002-11-08 2009-12-31 Ablynx N.V. Polypeptide constructs for nasal administration
US20100003253A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20100003248A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V Polypeptide constructs for rectal and/or vaginal administration
US20100003249A1 (en) * 2002-11-08 2010-01-07 Ablynx N.V. Polypeptide constructs for topical administration
US20100021459A1 (en) * 2002-11-08 2010-01-28 Ablynx N.V. Polypeptide constructs for intracellular delivery
US20100040613A1 (en) * 2002-11-08 2010-02-18 Ablynx N.V. Polypeptide constructs for sublingual administration
US9371381B2 (en) 2002-11-08 2016-06-21 Ablynx, N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US9725522B2 (en) 2002-11-08 2017-08-08 Ablynx N.V. Pulmonary administration of immunoglobulin single variable domains and constructs thereof
US20070237769A1 (en) * 2002-11-08 2007-10-11 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
US20110184150A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US20110184151A1 (en) * 2002-11-08 2011-07-28 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20110027281A1 (en) * 2002-11-08 2011-02-03 Ablynx N.V. Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US20110123529A1 (en) * 2002-11-08 2011-05-26 Ablynx N.V. Single domain antibodies directed against epidermal growth factor receptor and uses therefor
US20060034845A1 (en) * 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
US20110178277A1 (en) * 2002-11-08 2011-07-21 Ablynx N.V. Stabilized single domain antibodies
US20050260155A1 (en) * 2004-05-18 2005-11-24 Gopala Kovvali Compositions and methods for treatment of ulcerative colitis
US20080260757A1 (en) * 2004-06-01 2008-10-23 Domantis Limited Bispecific Fusion Antibodies With Enhanced Serum Half-Life
US8921528B2 (en) * 2004-06-01 2014-12-30 Domantis Limited Bispecific fusion antibodies with enhanced serum half-life
US9109229B2 (en) 2004-07-26 2015-08-18 Pfenex Inc. Process for improved protein expression by strain engineering
US8603824B2 (en) 2004-07-26 2013-12-10 Pfenex, Inc. Process for improved protein expression by strain engineering
US9067991B2 (en) 2005-05-18 2015-06-30 Ablynx N.V. Nanobodies against tumor necrosis factor-alpha
US11472871B2 (en) 2005-05-18 2022-10-18 Ablynx N.V. Nanobodies against tumor necrosis factor-alpha
US8188223B2 (en) 2005-05-18 2012-05-29 Ablynx N.V. Serum albumin binding proteins
US8703131B2 (en) 2005-05-21 2014-04-22 Ablynx N.V. Nanobodies against tumor necrosis factor-alpha
US20100297111A1 (en) * 2005-05-21 2010-11-25 Els Anna Alice Beirnaert Nanobodies against tumor necrosis factor-alpha
US20070269422A1 (en) * 2006-05-17 2007-11-22 Ablynx N.V. Serum albumin binding proteins with long half-lives
US10689640B2 (en) 2007-04-27 2020-06-23 Pfenex Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
US9394571B2 (en) 2007-04-27 2016-07-19 Pfenex Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
US9580719B2 (en) 2007-04-27 2017-02-28 Pfenex, Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
US20100137162A1 (en) * 2007-04-27 2010-06-03 Pfenex Biopharmaceuticals, Inc. Method for Rapidly Screening Microbial Hosts to Identify Certain Strains with Improved Yield and/or Quality in the Expression of Heterologous Proteins
US20080269070A1 (en) * 2007-04-27 2008-10-30 Dow Global Technologies, Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
EP2799448A1 (en) 2008-05-22 2014-11-05 Bristol-Myers Squibb Company Multivalent fibronectin based scaffold domain proteins
US9221918B2 (en) 2008-07-02 2015-12-29 Argen-X B.V. Antigen binding polypeptides
US9315576B2 (en) 2008-07-02 2016-04-19 Argen-X N.V. Antigen binding polypeptides
US20110300140A1 (en) * 2008-07-02 2011-12-08 Argen-X B.V. Antigen binding polypeptides
US9428580B2 (en) 2008-07-02 2016-08-30 Argen-X B.V. Antigen binding polypeptides
US9346891B2 (en) 2008-07-02 2016-05-24 Argen-X.N.V. Antigen binding polypeptides
US8444976B2 (en) 2008-07-02 2013-05-21 Argen-X B.V. Antigen binding polypeptides
US8524231B2 (en) * 2008-07-02 2013-09-03 Argen-X B.V. Antigen binding polypeptides
US20100330080A1 (en) * 2008-07-02 2010-12-30 Torsten Dreier Antigen binding polypeptides
US20100172894A1 (en) * 2008-10-29 2010-07-08 Wyeth Methods for purification of single domain antigen binding molecules
US9393304B2 (en) 2008-10-29 2016-07-19 Ablynx N.V. Formulations of single domain antigen binding molecules
US10118962B2 (en) 2008-10-29 2018-11-06 Ablynx N.V. Methods for purification of single domain antigen binding molecules
US9993552B2 (en) 2008-10-29 2018-06-12 Ablynx N.V. Formulations of single domain antigen binding molecules
US11370835B2 (en) 2008-10-29 2022-06-28 Ablynx N.V. Methods for purification of single domain antigen binding molecules
EP3029064A1 (en) 2008-11-24 2016-06-08 Bristol-Myers Squibb Company Bispecific egfr/igfir binding molecules
WO2010060095A1 (en) 2008-11-24 2010-05-27 Bristol-Myers Squibb Company Bispecific egfr/igfir binding molecules
WO2011051327A3 (en) * 2009-10-30 2011-06-30 Novartis Ag Small antibody-like single chain proteins
US9714281B2 (en) 2010-02-18 2017-07-25 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind IL-23
US8927693B2 (en) 2010-02-18 2015-01-06 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind IL-23
WO2011103105A1 (en) 2010-02-18 2011-08-25 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind il-23
WO2011130354A1 (en) 2010-04-13 2011-10-20 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind pcsk9
EP3424949A1 (en) 2010-04-13 2019-01-09 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind pcsk9
EP3091028A1 (en) 2010-05-26 2016-11-09 Bristol-Myers Squibb Company Fibronectin based scaffold proteins having improved stability
WO2011150133A2 (en) 2010-05-26 2011-12-01 Bristol-Myers Squibb Company Fibronectin based scaffold proteins having improved stability
US9775912B2 (en) 2010-11-26 2017-10-03 Molecular Partners Ag Designed repeat proteins binding to serum albumin
US9284361B2 (en) 2010-11-26 2016-03-15 Molecular Partners Ag Designed repeat proteins binding to serum albumin
WO2012088006A1 (en) 2010-12-22 2012-06-28 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind il-23
US9260496B2 (en) 2010-12-22 2016-02-16 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind IL-23
WO2013067029A2 (en) 2011-10-31 2013-05-10 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
WO2013119903A1 (en) 2012-02-10 2013-08-15 Research Corporation Technologies, Inc. Fusion proteins comprising immunoglobulin constant domain-derived scaffolds
WO2013138338A2 (en) 2012-03-12 2013-09-19 Massachusetts Institute Of Technology Methods for treating tissue damage associated with ischemia with apoliporotein d
WO2013177187A2 (en) 2012-05-22 2013-11-28 Massachusetts Institute Of Technology Synergistic tumor treatment with extended-pk il-2 and therapeutic agents
EP3835310A1 (en) 2012-09-13 2021-06-16 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind to myostatin
EP4397675A2 (en) 2012-09-13 2024-07-10 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind to myostatin
WO2014120891A2 (en) 2013-02-01 2014-08-07 Bristol-Myers Squibb Company Fibronectin based scaffold proteins
WO2014165093A2 (en) 2013-03-13 2014-10-09 Bristol-Myers Squibb Company Fibronectin based scaffold domains linked to serum albumin or a moiety binding thereto
WO2016025642A1 (en) 2014-08-12 2016-02-18 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2 and integrin-binding-fc-fusion protein
WO2016025645A1 (en) 2014-08-12 2016-02-18 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2, a therapeutic antibody, and an immune checkpoint blocker
EP3646879A1 (en) 2014-08-12 2020-05-06 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2 and integrin-binding-fc-fusion protein
WO2016025647A1 (en) 2014-08-12 2016-02-18 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2, a therapeutic antibody, and a cancer vaccine
EP3702367A1 (en) 2014-11-25 2020-09-02 Bristol-Myers Squibb Company Novel pd-l1 binding polypeptides for imaging
WO2016086021A1 (en) 2014-11-25 2016-06-02 Bristol-Myers Squibb Company Novel pd-l1 binding polypeptides for imaging
US10633438B2 (en) 2015-03-31 2020-04-28 Vhsquared Limited Polypeptides
US12234279B2 (en) 2015-03-31 2025-02-25 Sorriso Pharmaceuticals, Inc. Peptide construct having a protease-cleavable linker
US12173054B2 (en) 2015-03-31 2024-12-24 Sorriso Pharmaceuticals, Inc. Polypeptides
EP3985020A1 (en) 2015-04-24 2022-04-20 ViiV Healthcare UK (No.5) Limited Polypeptides targeting hiv fusion
WO2016171980A1 (en) 2015-04-24 2016-10-27 Bristol-Myers Squibb Company Polypeptides targeting hiv fusion
US12084518B2 (en) 2015-05-21 2024-09-10 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US9708412B2 (en) 2015-05-21 2017-07-18 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US10954311B2 (en) 2015-05-21 2021-03-23 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
EP3733698A1 (en) 2015-09-23 2020-11-04 Bristol-Myers Squibb Company Glypican-3 binding fibronectin based scafflold molecules
US10544221B2 (en) 2016-05-20 2020-01-28 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US9920115B2 (en) 2016-05-20 2018-03-20 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US11453716B2 (en) 2016-05-20 2022-09-27 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US10100106B2 (en) 2016-05-20 2018-10-16 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US12528859B2 (en) 2016-05-20 2026-01-20 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US10066016B2 (en) 2016-05-20 2018-09-04 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US11684677B2 (en) 2016-09-30 2023-06-27 Sorriso Pharmaceuticals, Inc. Compositions
US10844134B2 (en) 2016-11-23 2020-11-24 Harpoon Therapeutics, Inc. PSMA targeting trispecific proteins and methods of use
US10849973B2 (en) 2016-11-23 2020-12-01 Harpoon Therapeutics, Inc. Prostate specific membrane antigen binding protein
US10603358B2 (en) 2017-01-10 2020-03-31 Nodus Therapeutics Combination tumor treatment with an integrin-binding-Fc fusion protein and immune stimulator
US10350266B2 (en) 2017-01-10 2019-07-16 Nodus Therapeutics, Inc. Method of treating cancer with a multiple integrin binding Fc fusion protein
US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
US10730954B2 (en) 2017-05-12 2020-08-04 Harpoon Therapeutics, Inc. MSLN targeting trispecific proteins and methods of use
US10543271B2 (en) 2017-05-12 2020-01-28 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US11607453B2 (en) 2017-05-12 2023-03-21 Harpoon Therapeutics, Inc. Mesothelin binding proteins
WO2019036605A2 (en) 2017-08-17 2019-02-21 Massachusetts Institute Of Technology Multiple specificity binders of cxc chemokines and uses thereof
US10927180B2 (en) 2017-10-13 2021-02-23 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US11976125B2 (en) 2017-10-13 2024-05-07 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US12371504B2 (en) 2017-10-13 2025-07-29 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
WO2019154985A1 (en) 2018-02-12 2019-08-15 Biontech Rna Pharmaceuticals Gmbh Treatment using cytokine encoding rna
US12415860B2 (en) 2018-05-14 2025-09-16 Harpoon Therapeutics, Inc. Binding moiety for conditional activation of immunoglobulin molecules
WO2020020783A1 (en) 2018-07-24 2020-01-30 Biontech Rna Pharmaceuticals Gmbh Il2 agonists
US12195544B2 (en) 2018-09-21 2025-01-14 Harpoon Therapeutics, Inc. EGFR binding proteins and methods of use
US11807692B2 (en) 2018-09-25 2023-11-07 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use
US10815311B2 (en) 2018-09-25 2020-10-27 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use
WO2020068261A1 (en) 2018-09-28 2020-04-02 Massachusetts Institute Of Technology Collagen-localized immunomodulatory molecules and methods thereof
WO2020154032A1 (en) 2019-01-23 2020-07-30 Massachusetts Institute Of Technology Combination immunotherapy dosing regimen for immune checkpoint blockade
EP4414033A2 (en) 2019-02-08 2024-08-14 Biontech Cell & Gene Therapies Gmbh Treatment involving car-engineered t cells and cytokines
WO2020161224A1 (en) 2019-02-08 2020-08-13 Biontech Cell & Gene Therapies Gmbh Treatment involving car-engineered t cells and cytokines
WO2020187848A1 (en) 2019-03-18 2020-09-24 Biontech Cell & Gene Therapies Gmbh Lnterleukin-2 receptor (il2r) and interleukin-2 (il2) variants for specific activation of immune effector cells
WO2020200481A1 (en) 2019-04-05 2020-10-08 Biontech Rna Pharmaceuticals Gmbh Treatment involving interleukin-2 (il2) and interferon (ifn)
WO2020201448A1 (en) 2019-04-05 2020-10-08 Biontech Rna Pharmaceuticals Gmbh Treatment involving interleukin-2 (il2) and interferon (ifn)
US12516128B2 (en) 2019-05-14 2026-01-06 Harpoon Therapeutics, Inc. EpCAM binding proteins and methods of use
US12570736B2 (en) 2019-06-21 2026-03-10 Sorriso Pharmaceuticals, Inc. Compositions
US11667719B2 (en) 2019-06-21 2023-06-06 Sorriso Pharmaceuticals, Inc. VHH immunoglobulin chain variable domain that binds to IL-7R and methods of use thereof for treating autoimmune and/or inflammatory diseases
US12559552B2 (en) 2019-06-21 2026-02-24 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US12247079B2 (en) 2019-06-21 2025-03-11 Sorriso Pharmaceuticals, Inc. Polynucleotides encoding, and method of making, a polypeptide comprising a VHH which binds interleukin-receptor (IL-7R)
US11623952B2 (en) 2019-06-21 2023-04-11 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
WO2020260270A1 (en) 2019-06-24 2020-12-30 Biontech Rna Pharmaceuticals Gmbh Il2 agonists
WO2021058091A1 (en) 2019-09-24 2021-04-01 Biontech Rna Pharmaceuticals Gmbh Treatment involving therapeutic antibody and interleukin-2 (il2)
WO2021058472A1 (en) 2019-09-24 2021-04-01 Biontech Rna Pharmaceuticals Gmbh Treatment involving therapeutic antibody and interleukin-2 (il2)
WO2021127495A1 (en) 2019-12-20 2021-06-24 Regeneron Pharmaceuticals, Inc. Novel il2 agonists and methods of use thereof
WO2021127487A2 (en) 2019-12-20 2021-06-24 Regeneron Pharmaceuticals, Inc. Novel il2 agonists and methods of use thereof
WO2021130223A1 (en) 2019-12-23 2021-07-01 Biontech Cell & Gene Therapies Gmbh Treatment involving immune effector cells genetically modified to express antigen receptors
WO2021130225A1 (en) 2019-12-27 2021-07-01 Biontech Cell & Gene Therapies Gmbh In vitro and in vivo gene delivery to immune effector cells using nanoparticles functionalized with designed ankyrin repeat proteins (darpins)
US11180563B2 (en) 2020-02-21 2021-11-23 Harpoon Therapeutics, Inc. FLT3 binding proteins and methods of use
WO2021185775A1 (en) 2020-03-16 2021-09-23 Biontech Cell & Gene Therapies Gmbh Antigen-specific t cell receptors and t cell epitopes
WO2021198258A1 (en) 2020-03-31 2021-10-07 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination
WO2021197589A1 (en) 2020-03-31 2021-10-07 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination
WO2021231447A1 (en) 2020-05-12 2021-11-18 Regeneron Pharmaceuticals, Inc. Novel il10 agonists and methods of use thereof
WO2022136257A1 (en) 2020-12-21 2022-06-30 BioNTech SE Therapeutic rna for treating cancer
WO2022135667A1 (en) 2020-12-21 2022-06-30 BioNTech SE Therapeutic rna for treating cancer
WO2022136266A1 (en) 2020-12-21 2022-06-30 BioNTech SE Therapeutic rna for treating cancer
WO2022136255A1 (en) 2020-12-21 2022-06-30 BioNTech SE Treatment schedule for cytokine proteins
WO2022135666A1 (en) 2020-12-21 2022-06-30 BioNTech SE Treatment schedule for cytokine proteins
WO2022218891A2 (en) 2021-04-12 2022-10-20 BioNTech SE Rna compositions comprising a buffer substance and methods for preparing, storing and using the same
WO2022223617A1 (en) 2021-04-20 2022-10-27 BioNTech SE Virus vaccine
WO2023004282A2 (en) 2021-07-19 2023-01-26 Regeneron Pharmaceuticals, Inc. Il12 receptor agonists and methods of use thereof
WO2023022965A2 (en) 2021-08-16 2023-02-23 Regeneron Pharmaceuticals, Inc. Novel il27 receptor agonists and methods of use thereof
WO2023051926A1 (en) 2021-09-30 2023-04-06 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination and pd-1 axis binding antagonists
WO2023052531A1 (en) 2021-09-30 2023-04-06 BioNTech SE Treatment involving non-immunogenic rna for antigen vaccination and pd-1 axis binding antagonists
WO2023066496A1 (en) 2021-10-21 2023-04-27 BioNTech SE Coronavirus vaccine
EP4238577A2 (en) 2021-10-22 2023-09-06 BioNTech SE Compositions for administration of different doses of rna
WO2023067193A2 (en) 2021-10-22 2023-04-27 BioNTech SE Compositions for administration of different doses of rna
WO2023083916A1 (en) 2021-11-09 2023-05-19 BioNTech SE Rna encoding peptidoglycan hydrolase and use thereof for treating bacterial infection
WO2023083434A1 (en) 2021-11-09 2023-05-19 BioNTech SE Rna encoding peptidoglycan hydrolase and use thereof for treating bacterial infection
WO2023086812A1 (en) 2021-11-11 2023-05-19 Regeneron Pharmaceuticals, Inc. Cd20-pd1 binding molecules and methods of use thereof
WO2023126053A1 (en) 2021-12-28 2023-07-06 BioNTech SE Lipid-based formulations for administration of rna
WO2023126404A1 (en) 2021-12-28 2023-07-06 BioNTech SE Lipid-based formulations for administration of rna
WO2023166099A1 (en) 2022-03-01 2023-09-07 BioNTech SE RNA LIPID NANOPARTICLES (LNPs) COMPRISING A POLYOXAZOLINE AND/OR POLYOXAZINE POLYMER
WO2023193892A1 (en) 2022-04-05 2023-10-12 BioNTech SE Nucleic acid compositions comprising an inorganic polyphosphate and methods for preparing, storing and using the same
WO2023194508A1 (en) 2022-04-05 2023-10-12 BioNTech SE Nucleic acid compositions comprising a multivalent anion, such as an inorganic polyphosphate, and methods for preparing, storing and using the same
WO2023215810A1 (en) * 2022-05-05 2023-11-09 Inhibrx, Inc. Albumin-binding polypeptides and uses thereof
WO2024002985A1 (en) 2022-06-26 2024-01-04 BioNTech SE Coronavirus vaccine
WO2024018035A1 (en) 2022-07-21 2024-01-25 BioNTech SE Multifunctional cells transiently expressing an immune receptor and one or more cytokines, their use and methods for their production
WO2024017479A1 (en) 2022-07-21 2024-01-25 BioNTech SE Multifunctional cells transiently expressing an immune receptor and one or more cytokines, their use and methods for their production
WO2024028325A1 (en) 2022-08-01 2024-02-08 BioNTech SE Nucleic acid compositions comprising amphiphilic oligo ethylene glycol (oeg)-conjugated compounds and methods of using such compounds and compositions
WO2024027910A1 (en) 2022-08-03 2024-02-08 BioNTech SE Rna for preventing or treating tuberculosis
WO2024028445A1 (en) 2022-08-03 2024-02-08 BioNTech SE Rna for preventing or treating tuberculosis
WO2024151978A1 (en) 2023-01-13 2024-07-18 Regeneron Pharmaceuticals, Inc. Il12 receptor agonists and methods of use thereof
WO2024153324A1 (en) 2023-01-18 2024-07-25 BioNTech SE Rna formulations for pharmaceutical use
WO2024153675A1 (en) 2023-01-18 2024-07-25 BioNTech SE Rna formulations for pharmaceutical use
WO2024213776A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024216214A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024216212A1 (en) 2023-04-14 2024-10-17 BioNTech SE Rna for preventing or treating tuberculosis
WO2024233926A1 (en) 2023-05-10 2024-11-14 Regeneron Pharmaceuticals, Inc. Cd20-pd1 binding molecules and methods of use thereof
WO2025006676A2 (en) 2023-06-27 2025-01-02 Firecyte Therapeutics, Inc. Insulin-like growth factor binding protein like 1 (igfbpl1) compositions and methods of use thereof
US12234297B2 (en) 2023-07-13 2025-02-25 Phylaxis Bioscience, Llc IgE binding proteins and uses thereof
WO2025021668A1 (en) 2023-07-21 2025-01-30 BioNTech SE Agents and methods for targeted delivery of cytokines to immune cells
WO2025026866A1 (en) 2023-07-28 2025-02-06 BioNTech SE Rna therapeutics with reduced toxicity
WO2025101851A1 (en) 2023-11-10 2025-05-15 Regeneron Pharmaceuticals, Inc. Engineered alpha klotho polypeptides and uses thereof
WO2025106754A1 (en) 2023-11-15 2025-05-22 BioNTech SE Coronavirus vaccine
WO2025255508A1 (en) 2024-06-07 2025-12-11 Regeneron Pharmaceuticals, Inc. Engineered alpha klotho polypeptides and uses thereof
WO2026082899A2 (en) 2024-10-16 2026-04-23 BioNTech SE Rna for preventing or treating tuberculosis

Also Published As

Publication number Publication date
RU2357974C2 (en) 2009-06-10
RU2012108416A (en) 2013-09-10
BRPI0406694B8 (en) 2021-05-25
JP2006524036A (en) 2006-10-26
CA2512545A1 (en) 2004-07-29
JP2006519763A (en) 2006-08-31
NO20053774L (en) 2005-08-31
US20100021459A1 (en) 2010-01-28
CA2505326A1 (en) 2004-05-21
US9028816B2 (en) 2015-05-12
JP2006517789A (en) 2006-08-03
NO337265B1 (en) 2016-02-29
WO2004062551A2 (en) 2004-07-29
NZ540194A (en) 2008-07-31
KR20080113286A (en) 2008-12-29
EP1587838A2 (en) 2005-10-26
JP2006520584A (en) 2006-09-14
JP2011024581A (en) 2011-02-10
WO2004041865A3 (en) 2004-07-15
EP2316852A1 (en) 2011-05-04
ZA200504996B (en) 2008-02-27
WO2004041865A2 (en) 2004-05-21
US20100040613A1 (en) 2010-02-18
NZ540196A (en) 2008-09-26
US20060115470A1 (en) 2006-06-01
US20190112363A1 (en) 2019-04-18
CA2512545C (en) 2015-06-30
EP2390270A1 (en) 2011-11-30
BRPI0406694B1 (en) 2017-03-07
CN101412759A (en) 2009-04-22
ES2655912T3 (en) 2018-02-22
EP2390268A1 (en) 2011-11-30
CA2505316C (en) 2014-08-05
ES2551682T3 (en) 2015-11-23
NZ540771A (en) 2009-05-31
ATE517922T1 (en) 2011-08-15
CA2505325A1 (en) 2004-05-21
CN102584997A (en) 2012-07-18
NZ540195A (en) 2009-01-31
US20110184145A1 (en) 2011-07-28
EP2284192A2 (en) 2011-02-16
US20090324512A1 (en) 2009-12-31
CN1735630A (en) 2006-02-15
HK1082745A1 (en) 2006-06-16
CA2505316A1 (en) 2004-05-21
CA2505325C (en) 2014-02-25
CN100439400C (en) 2008-12-03
EP2316852B1 (en) 2014-03-05
MXPA05004955A (en) 2005-11-17
ES2542330T3 (en) 2015-08-04
EP1900753A3 (en) 2008-04-09
EP2301967A2 (en) 2011-03-30
EP2336179A1 (en) 2011-06-22
AU2004204262A1 (en) 2004-07-29
ES2466716T3 (en) 2014-06-11
RU2005125430A (en) 2006-02-10
MXPA05006043A (en) 2006-01-30
US20100003248A1 (en) 2010-01-07
AU2004204262B2 (en) 2010-11-04
AU2010226938A1 (en) 2010-10-28
BRPI0406694A (en) 2005-12-20
EP1900753B1 (en) 2017-08-09
KR20050092029A (en) 2005-09-16
US20150299301A1 (en) 2015-10-22
EP2301967A3 (en) 2011-07-13
US10112989B2 (en) 2018-10-30
US11034755B2 (en) 2021-06-15
EP1587838B1 (en) 2015-04-15
WO2004041863A3 (en) 2004-07-15
NZ563471A (en) 2009-04-30
US9243065B2 (en) 2016-01-26
IL168357A (en) 2011-09-27
AU2010226938B2 (en) 2012-03-29
EP2390268B1 (en) 2017-11-01
DK2316852T3 (en) 2014-06-16
US20100003249A1 (en) 2010-01-07
EP2284192A3 (en) 2011-07-20
US20060149041A1 (en) 2006-07-06
WO2004041863A2 (en) 2004-05-21
WO2004062551A3 (en) 2004-09-02
PT2316852E (en) 2014-06-23
WO2004041862A2 (en) 2004-05-21
EP1900753A2 (en) 2008-03-19
EP3299393A1 (en) 2018-03-28
EP2258392A1 (en) 2010-12-08
RU2704232C2 (en) 2019-10-24
WO2004041862A3 (en) 2004-06-24

Similar Documents

Publication Publication Date Title
EP2316852B1 (en) Stabilized single domain antibodies
AU2003286003B2 (en) Stabilized single domain antibodies
US9371381B2 (en) Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor
US20060034833A1 (en) Single domain antibodies directed against interferron-gamma and uses therefor
CN1735629B (en) Single domain antibodies against tumor necrosis factor-alpha and uses thereof
HK1162539A (en) Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
HK1082745B (en) Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
HK1162539B (en) Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor

Legal Events

Date Code Title Description
AS Assignment

Owner name: ABLYNX N.V., BELGIUM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SILENCE, KAREN;LAUWEREYS, MARC JOZEF;DREIER, TORSTEN;REEL/FRAME:022592/0169;SIGNING DATES FROM 20080225 TO 20090304

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION