US20100303821A1 - Immunoglobulins - Google Patents

Immunoglobulins Download PDF

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US20100303821A1
US20100303821A1 US12/787,588 US78758810A US2010303821A1 US 20100303821 A1 US20100303821 A1 US 20100303821A1 US 78758810 A US78758810 A US 78758810A US 2010303821 A1 US2010303821 A1 US 2010303821A1
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seq
asthma
antigen binding
binding protein
antibody
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Claire Ashman
Jonathan Henry Ellis
Paul Andrew Hamblin
Alan Peter Lewis
Martin Anibal Orecchia
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Glaxo Group Ltd
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Glaxo Group Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61P1/16—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
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • 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
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A61P33/12—Schistosomicides
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/247—IL-4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/50—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to antigen binding proteins, particularly antibodies that bind to interleukin 13 (IL-13) and neutralise the activity thereof, polynucleotides encoding such antigen binding proteins, pharmaceutical formulations containing said antigen binding proteins and to the use of such antigen binding proteins in the treatment and/or prophylaxis of diseases associated with inflammation, such as asthma.
  • IL-13 interleukin 13
  • Interleukin-13 (IL-13)
  • IL-13 is a 12 kDa secreted cytokine originally described as a T cell-derived cytokine that inhibits inflammatory cytokine production. Structural studies indicate that it has a four-helical bundle arrangement held by two disulphide bonds. Although IL-13 has four potential glycosylation sites, analysis of native IL-13 from rat lung has indicated that it is produced as an unglycosylated molecule. Expression of human IL-13 from NSO and COS-7 cells confirms this observation (Eisenmesser et al, J. Mol. Biol. 2001 310(1):231-241; Moy et al, J. Mol. Biol. 2001 310(1):219-230; Cannon-Carlson et al, Protein Expression and Purification 1998 12(2):239-248).
  • IL-13 has been implicated in asthma, Chronic Obstructive Pulmonary Disease (COPD), Allergic disease including atopic dermatitis and allergic rhinitis, Esophagal eosinophilia, Oncology Indications, e.g. B-cell chronic lymphocytic leukemia (B-CLL) and Hodgkin's disease, Inflammatory Bowel Diseases e.g. ulcerative colitis, Crohn's disease and indeterminate colitis, Psoriasis and Psoriatic Arthritis, Acute graft-versus-host disease, Diabetic nephropathy, Fibrotic Conditions such as Pulmonary fibrosis e.g. Idiopathic Pulmonary Fibrosis (IPF).
  • COPD Chronic Obstructive Pulmonary Disease
  • IPF Idiopathic Pulmonary Fibrosis
  • the invention provides antigen binding proteins which bind to IL-13, for example IL-13 antibodies, and to the combination of such IL-13 antibodies with an IL-4 antagonist and/or an IL-5 antagonist.
  • the IL-13 antibodies of the present invention are related to, or derived from, a murine mAb 6A1, wherein the CDRH3 is mutated.
  • the 6A1 murine heavy chain variable region amino acid sequence is provided as SEQ ID NO: 58.
  • the 6A1 murine light chain variable region amino acid sequence is provided as SEQ ID NO 59.
  • the heavy chain variable regions (VH) of the present invention comprise the following CDRs (as defined by Kabat (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987)):
  • the CDRs of the heavy chain variable regions of the present invention may comprise the following CDRs:
  • H1 DTYMH H2 TIDPANGNTKYVPKFQG
  • H3 WIYDDYHYDDYYAMDY SEQ ID NO: 4
  • SVYDDYHYDDYYAMDY SEQ ID NO: 5
  • SIFDDYHYDDYYAMDY SEQ ID NO: 6
  • SIYEDYHYDDYYAMDY SEQ ID NO: 7
  • SIYDDYAYDDYYAMDY SEQ ID NO: 8
  • SIYDDYEYDDYYAMDY SIYDDYEYDDYYAMDY
  • SIYDDYQYDDYYAMDY SEQ ID NO: 10
  • SIYDDYRYDDYYAMDY SEQ ID NO: 11
  • SIYDDYSYDDYYAMDY SEQ ID NO: 12
  • SIYDDYTYDDYYAMDY SEQ ID NO: 13
  • SIYDDYVYDDYYAMDY SEQ ID NO: 14
  • the light chain variable regions of the present invention comprise the following CDRs (as defined by Kabat):
  • the CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987), as set out in the tables above, alternatively they can be determined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927-948), the contact definition method (MacCallum R. M., and Martin A. C. R. and Thornton J. M, (1996), Journal of Molecular Biology, 262 (5), 732-745) or any other established method for numbering the residues in an antibody and determining CDRs known to the skilled man in the art.
  • CDRH1 may be defined as comprising FYIKDTYMH (SEQ ID NO 60) or GFYIKDTYMH (SEQ ID NO 61).
  • the present invention also provides an antigen-binding protein comprising the IL-13 antibody of the present invention which is linked to one or more epitope-binding domains, for example an antigen-binding protein comprising the IL-13 antibody of the present invention linked to an epitope-binding domain which is capable of binding to IL-4, or an antigen-binding protein comprising the IL-13 antibody of the present invention linked to an epitope-binding domain which is capable of binding to IL-5, or an antigen-binding protein comprising the IL-13 antibody of the present invention linked to a first epitope-binding domain which is capable of binding to IL-4 and a second epitope-binding domain which is capable of binding to IL-5.
  • the present invention also provides a method of decreasing the aggregation propensity of an immunoglobulin single variable domain, for example a human VK domain antibody, by mutation of residue 89 (kabat numbering) to ‘Q’ (glutamine).
  • this method can be applied to the anti-IL-4 domain antibody of SEQ ID NO: 80, resulting in a mutated dAb sequence, for example SEQ ID NO:94.
  • Such mutated dAbs may be alone or as part of a larger sequence, for example part of a mAbdAb sequence, resulting in for example, a dAb comprising a sequence selected from SEQ ID NO: 117-134.
  • the invention also provides human VK dAbs which have improved aggregation profiles, for example a human VK dAb derived from a germline framework selected from IGKV1-17, IGKV1D-17, IGKV1/OR2-108, IGKV1-6, IGKV5-2, IGKV1D-42, IGKV2-24, IGKV2-28, IGKV2-30, IGKV2-40, IGKV2D-29, IGKV2D-30, IGKV2D-24 and IGKV6-21 wherein residue 89 (kabat numbering) of the VK dAb is ‘Q’ (glutamine).
  • a human VK dAb derived from a germline framework selected from IGKV1-17, IGKV1D-17, IGKV1/OR2-108, IGKV1-6, IGKV5-2, IGKV1D-42, IGKV2-24, IGKV2-28, IGKV2-30,
  • the VK dAb comprises germline framework regions selected from the germline frameworks of IGKV1-17, IGKV1D-17, IGKV1/OR2-108, IGKV1-6, IGKV5-2, IGKV1D-42, IGKV2-24, IGKV2-28, IGKV2-30, IGKV2-40, IGKV2D-29, IGKV2D-30, IGKV2D-24 and IGKV6-21 wherein residue 89 (kabat numbering) of the VK dAb is ‘Q’ (glutamine).
  • the invention provides a human dAb comprising the sequence of SEQ ID NO: 94.
  • the invention also provides a polynucleotide sequence encoding a heavy chain of any of the antigen-binding proteins described herein, and a polynucleotide encoding a light chain of any of the antigen-binding proteins described herein.
  • Such polynucleotides represent the coding sequence which corresponds to the equivalent polypeptide sequences, however it will be understood that such polynucleotide sequences could be cloned into an expression vector along with a start codon, an appropriate signal sequence and a stop codon.
  • the invention also provides a recombinant transformed or transfected host cell comprising one or more polynucleotides encoding a heavy chain and a light chain of any of the antigen-binding proteins described herein.
  • the invention further provides a method for the production of any of the antigen-binding proteins described herein which method comprises the step of culturing a host cell comprising a first and second vector, said first vector comprising a polynucleotide encoding a heavy chain of any of the antigen-binding proteins described herein and said second vector comprising a polynucleotide encoding a light chain of any of the antigen-binding proteins described herein, in a suitable culture media, for example serum-free culture media.
  • a suitable culture media for example serum-free culture media.
  • the invention further provides a pharmaceutical composition comprising an antigen-binding protein as described herein a pharmaceutically acceptable carrier.
  • the present invention provides a method of treatment or prophylaxis of diseases or disorders associated with atopic diseases/disorders and chronic inflammatory diseases/disorders by administration of the antigen binding protein of the present invention.
  • asthma such as allergic asthma, particularly severe asthma (that is asthma that is unresponsive to current treatment, including systemically administered corticosteroids; see Busse W W et al, J. Allergy Clin.
  • asthma defined as the asthmatic phenotype characterised by failure to achieve control despite maximally recommended doses of prescribed inhaled steroids, see Barnes P J (1998), Eur Respir J 12:1208-1218
  • brittle defined asthma (defines a subgroup of patients with severe, unstable asthma who maintain a wide peak expiratory flow (PEF) variability despite high doses of inhaled steroids, see Ayres J G et al (1998) Thorax 58:315-321), nocturnal asthma, premenstrual asthma, steroid resistant asthma (see Woodcock A J (1993) Eur Respir J 6:743-747), steroid dependent asthma (defined as asthma that can be controlled only with high doses of oral steroids), aspirin induced asthma, adult-onset asthma, paediatric asthma.
  • Antibodies of the invention may be used to prevent, reduce the frequency of, or mitigate the effects of acute, asthmatic episodes (status asthmaticus). Antibodies of the invention may also be used to reduce the dosing required (either in terms of amount administered or frequency of dosing) of other medicaments used in the treatment of asthma. For example, antibodies of the invention may be used to reduce the dosing required for steroid treatment of asthma such as corticosteroid treatment (“steroid sparing”).
  • diseases or disorders that may be treated with antibodies of the invention include atopic dermatitis, allergic rhinitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, and diseases of cell cycle regulation, e.g. Hodgkins disease, B cell chronic lymphocytic leukaemia.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis,
  • the invention provides the use of an antigen binding protein of the invention in the preparation of a medicament for treatment or prophylaxis of atopic diseases/disorders and chronic inflammatory diseases/disorders.
  • asthma such as allergic asthma, particularly severe asthma (that is asthma that is unresponsive to current treatment, including systemically administered corticosteroids; see Busse W W et al, J. Allergy Clin.
  • asthma defined as the asthmatic phenotype characterised by failure to achieve control despite maximally recommended doses of prescribed inhaled steroids, see Barnes P J (1998), Eur Respir J 12:1208-1218
  • brittle defined asthma (defines a subgroup of patients with severe, unstable asthma who maintain a wide peak expiratory flow (PEF) variability despite high doses of inhaled steroids, see Ayres J G et al (1998) Thorax 58:315-321), nocturnal asthma, premenstrual asthma, steroid resistant asthma (see Woodcock A J (1993) Eur Respir J 6:743-747), steroid dependent asthma (defined as asthma that can be controlled only with high doses of oral steroids), aspirin induced asthma, adult-onset asthma, paediatric asthma.
  • Antibodies of the invention may be used to prevent, reduce the frequency of, or mitigate the effects of acute, asthmatic episodes (status asthmaticus). Antibodies of the invention may also be used to reduce the dosing required (either in terms of amount administered or frequency of dosing) of other medicaments used in the treatment of asthma. For example, antibodies of the invention may be used to reduce the dosing required for steroid treatment of asthma such as corticosteroid treatment (“steroid sparing”).
  • diseases or disorders that may be treated with antibodies of the invention include atopic dermatitis, allergic rhinitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, and diseases of cell cycle regulation, e.g. Hodgkins disease, B cell chronic lymphocytic leukaemia.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis,
  • binds to human IL-13 as used throughout the present specification in relation to antigen binding proteins thereof of the invention means that the antigen binding protein binds human IL-13 (hereinafter referred to as hIL-13) with no or insignificant binding to other human proteins such as IL-4.
  • the antigen binding proteins of the present invention bind to human IL-13 in that they can be seen to bind to human IL-13 in a Biacore assay (for example the Biacore assay described in example 3).
  • Biacore assay for example the Biacore assay described in example 3.
  • antigen binding protein refers to antibodies, antibody fragments and other protein constructs which are capable of binding to and neutralising human IL-13.
  • Fv, Fc, Fd, Fab, or F(ab) 2 are used with their standard meanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).
  • a “chimeric antibody” refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.
  • a “humanised antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)).
  • a suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT® database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • the prior art describes several ways of producing such humanised antibodies—see for example EP-A-0239400 and EP-A-054951
  • donor antibody refers to an antibody (monoclonal, and/or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
  • acceptor antibody refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
  • a human antibody is the acceptor antibody.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate).
  • the structure and protein folding of the antibody may mean that other residues are considered part of the antigen binding region and would be understood to be so by a skilled person. See for example Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.
  • domain refers to a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • An “antibody single variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • immunoglobulin single variable domain refers to an antibody variable domain (V H , V HH , V L ) that specifically binds an antigen or epitope independently of a different V region or domain.
  • An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other, different variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains).
  • a “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” which is capable of binding to an antigen as the term is used herein.
  • An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V HH dAbs (nanobodies).
  • Camelid V HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • Such V HH domains may be humanised according to standard techniques available in the art, and such domains are still considered to be “domain antibodies” according to the invention.
  • V H includes camelid V HH domains.
  • NARV are another type of immunoglobulin single variable domain which were identified in cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen Receptor variable region (commonly abbreviated to V(NAR) or NARV). For further details see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.
  • Epitope-binding domain refers to a domain that specifically binds an antigen or epitope independently of a different V region or domain, this may be a domain antibody (dAb), for example a human, camelid or shark immunoglobulin single variable domain or it may be a domain which is a derivative of a non-Immunoglobulin scaffold, for example a non-immunoglobulin scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human ⁇ -crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human prote
  • CTLA-4 Cytotoxic T Lymphocyte-associated Antigen 4
  • CTLA-4 is a CD28-family receptor expressed on mainly CD4+ T-cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties.
  • CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies. For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001)
  • Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid ⁇ -sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 and US20070224633
  • An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen.
  • the domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomisation of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1
  • Avimers are multidomain proteins derived from the A-domain scaffold family.
  • the native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007)
  • a transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem. 274, 24066-24073 (1999).
  • DARPins Designed Ankyrin Repeat Proteins
  • Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton.
  • a single ankyrin repeat is a 33 residue motif consisting of two ⁇ -helices and a ⁇ -turn. They can be engineered to bind different target antigens by randomising residues in the first ⁇ -helix and a ⁇ -turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation).
  • affinity maturation For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.
  • Fibronectin is a scaffold which can be engineered to bind to antigen.
  • Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the ⁇ -sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.
  • Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site.
  • TrxA thioredoxin
  • Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges—examples of microproteins include KalataB1 and conotoxin and knottins.
  • the microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein.
  • knottin domains see WO2008098796.
  • epitope binding domains include proteins which have been used as a scaffold to engineer different target antigen binding properties include human ⁇ -crystallin and human ubiquitin (affilins), kunitz type domains of human protease inhibitors, PDZ-domains of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7—Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). Epitope binding domains of the present invention could be derived from any of these alternative protein domains.
  • the term “antigen-binding site” refers to a site on a protein which is capable of specifically binding to antigen, this may be a single domain, for example an epitope-binding domain, or it may be paired VH/VL domains as can be found on a standard antibody.
  • single-chain Fv (ScFv) domains can provide antigen-binding sites.
  • mAbdAb and dAbmAb are used herein to refer to antigen-binding proteins of the present invention.
  • the two terms can be used interchangeably, and are intended to have the same meaning as used herein.
  • antigen binding protein refers to antibodies, antibody fragments, for example a domain antibody (dAb), ScFv, FAb, FAb 2 , and other protein constructs which are capable of binding to IL-13.
  • Antigen binding molecules may comprise at least one Ig variable domain, for example antibodies, domain antibodies, Fab, Fab′, F(ab′) 2 , Fv, ScFv, diabodies, mAbdAbs, affibodies, heteroconjugate antibodies or bispecifics.
  • the antigen binding molecule is an antibody.
  • the antigen binding molecule is a dAb, i.e.
  • Antigen binding molecules may be capable of binding to two targets, I.e. they may be dual targeting proteins.
  • Antigen binding molecules may be a combination of antibodies and antigen binding fragments such as for example, one or more domain antibodies and/or one or more ScFvs linked to a monoclonal antibody.
  • Antigen binding molecules may also comprise a non-Ig domain for example a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human ⁇ -crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to IL-13.
  • a non-Ig domain for example a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin;
  • antigen binding protein will be capable of antagonising and/or neutralising human IL-13.
  • an antigen binding protein may block IL-13 activity by binding to IL-13 and preventing a natural ligand from binding and/or activating the receptor.
  • IL-13 antagonist includes any compound capable of reducing and or eliminating at least one activity of IL-13.
  • an IL-13 antagonist may bind to IL-13 and that binding may directly reduce or eliminate IL-13 activity or it may work indirectly by blocking at least one ligand from binding the receptor.
  • IL-4 antagonist includes any compound capable of reducing and or eliminating at least one activity of IL-4.
  • an IL-4 antagonist may bind to IL-4 and that binding may directly reduce or eliminate IL-4 activity or it may work indirectly by blocking at least one ligand from binding the receptor.
  • IL-5 antagonist includes any compound capable of reducing and or eliminating at least one activity of IL-5.
  • an IL-5 antagonist may bind to IL-5 and that binding may directly reduce or eliminate IL-5 activity or it may work indirectly by blocking at least one ligand from binding the receptor.
  • the antigen binding proteins of the present invention comprise a heavy chain variable region containing a CDRH3 selected from the list consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14 and SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and a suitable CDRH1 and CDRH2, paired with a light chain variable region containing a suitable CDRL1, CDRL2 and CDRL3 to form an antigen binding Fv unit which binds to human IL-13.
  • the antigen binding proteins of the present invention neutralise the activity of human IL-13
  • the CDRH1 as set out in SEQ ID NO: 1 or SEQ ID NO: 60, or SEQ ID NO:61 and CDRH2 as set out in SEQ ID NO: 2 are also present in the heavy chain variable region.
  • the CDRHL1 as set out in SEQ ID NO: 19, CDRL2 as set out in SEQ ID NO:20 and CDRL3 as set out in SEQ ID NO: 21 are also present in the light chain variable region.
  • the antigen binding protein binds to human IL-13 with high affinity as measured by Biacore of 10 nM or less, and more particularly 2 nM or less, for example between about 0.8 nM and 2 nM, 1 nM or less, or 100 pM or less, for example between about 20 pM and about 100 pM or between about 20 pM and about 80 pM, or between about 20 pM and about 60 pM. In one such embodiment, this is measured by Biacore with the antigen binding protein being captured on the biosensor chip, for example as set out in Example 3.
  • the heavy chain variable regions of the present invention may be formatted together with light chain variable regions to allow binding to human IL-13, in the conventional immunoglobulin manner (for example, human IgG, IgA, IgM etc.) or in any other “antibody-like” format that binds to human IL-13 (for example, single chain Fv, diabodies, TandabsTM etc (for a summary of alternative “antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136)).
  • the antigen binding proteins of the present invention are derived from the murine antibody having the variable regions as described in SEQ ID NO:58 and SEQ ID NO:59 or non-murine equivalents thereof, such as rat, human, chimeric or humanised variants thereof, for example they are derived from the humanised antibody having the heavy and light chains as described in SEQ ID NO:22 and SEQ ID NO:24.
  • an antigen binding protein for example an antibody which binds human IL-13 and which comprises variants of the CDRH3 SIYDDYHYDDYYAMDY (SEQ ID NO: 3), wherein CDRH3 is substituted by the alternative amino acids set out below at one or more of the following positions (using Kabat numbering):
  • an antigen binding protein for example an antibody which binds human IL-13 and which comprises the CDRH3 set out in SEQ ID NO: 3, wherein CDRH3 comprises one or more of the following substitutions: S95W, I96V, Y97F, D98E, H100A_A, H100A_E, H100A_Q, H100A_R, H100A_S, H100A_T, H100A_V, Y100B_A, Y100B_I, Y100B_W, and Y100B_V.
  • the antigen binding protein of the present invention for example the antibody of the present invention, comprises a CDRH3 sequence selected from those set out in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14 and SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • Such antigen binding proteins may further comprise the following CDR sequences:
  • CDRH1 selected from SEQ ID NO:1, 60 and 61,
  • CDRH2 SEQ ID NO: 2;
  • CDRL1 SEQ ID NO:19;
  • CDRL2 SEQ ID NO:20;
  • CDRL3 SEQ ID NO:21.
  • CDRH2 SEQ ID NO:2;
  • CDRL1 SEQ ID NO:19;
  • CDRL2 SEQ ID NO:20;
  • CDRL3 SEQ ID NO:21.
  • CDRH2 SEQ ID NO:2;
  • CDRL1 SEQ ID NO:19;
  • CDRL2 SEQ ID NO:20;
  • CDRL3 SEQ ID NO:21.
  • CDRH2 SEQ ID NO:2;
  • CDRL1 SEQ ID NO:19;
  • CDRL2 SEQ ID NO:20;
  • CDRL3 SEQ ID NO:21.
  • CDRH2 SEQ ID NO:2;
  • CDRL1 SEQ ID NO:19;
  • CDRL2 SEQ ID NO:20;
  • CDRL3 SEQ ID NO:21.
  • an antigen binding protein such as a humanised antibody or antigen binding fragment thereof, comprising a heavy chain having the sequence selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; and the light chain of SEQ ID NO:24.
  • the invention provides an antigen binding protein, such as a humanised antibody or antigen binding fragment thereof, comprising a heavy chain selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90.
  • a humanised antibody or antigen binding fragment thereof comprising a heavy chain selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID
  • the invention also provides an antigen binding protein, such as a humanised antibody or antigen binding fragment thereof, comprising a light chain selected from SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 and SEQ ID NO: 114.
  • an antigen binding protein such as a humanised antibody or antigen binding fragment thereof, comprising a light chain selected from SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 and SEQ ID NO: 114.
  • the invention further provides an antigen binding protein, such as a humanised antibody or antigen binding fragment thereof, comprising a heavy chain having the sequence selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; and the light chain of SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 and SEQ ID NO: 114.
  • an antigen binding protein such as a humanised antibody or antigen binding fragment thereof, comprising a heavy chain having the sequence selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
  • the antigen binding protein of the present invention comprises an antibody comprising a heavy chain selected from SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91 and a light chain selected from SEQ ID NO:24, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 and SEQ ID NO: 114.
  • the antigen binding protein of the present invention comprises a heavy chain selected from SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54; and a light chain selected from SEQ ID NO: 24, SEQ ID NO: 108, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114, for example the antigen binding protein comprises the heavy chain of SEQ ID NO: 48 and the light chain of SEQ ID NO:24, or the heavy chain of SEQ ID NO: 50 and the light chain of SEQ ID NO:24, or the heavy chain of SEQ ID NO: 52 and the light chain of SEQ ID NO:24, or the heavy chain of SEQ ID NO: 54 and the light chain of SEQ ID NO:24, or the heavy chain of SEQ ID NO: 88 and the light chain of SEQ ID NO:24 or the heavy chain of SEQ ID NO: 89 and the light chain of SEQ ID NO:24, or the heavy chain of SEQ ID NO: 90 and the light chain of SEQ ID NO:
  • the antigen binding protein of the present invention comprises a heavy chain selected from SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54; and a light chain selected from SEQ ID NO:108 and SEQ ID NO:110, for example the antigen binding protein comprises the heavy chain of SEQ ID NO: 48 and the light chain of SEQ ID NO:108, or the antigen binding protein comprises the heavy chain of SEQ ID NO: 48 and the light chain of SEQ ID NO:110, or the antigen binding protein comprises the heavy chain of SEQ ID NO: 50 and the light chain of SEQ ID NO:108, or the antigen binding protein comprises the heavy chain of SEQ ID NO: 50 and the light chain of SEQ ID NO:110, or the antigen binding protein comprises the heavy chain of SEQ ID NO: 52 and the light chain of SEQ ID NO:108, or the antigen binding protein comprises the heavy chain of SEQ ID NO: 52 and the light chain of SEQ ID NO:110, or the antigen binding protein comprises the heavy chain of
  • the IL-13 antibodies of the present invention may be combined with an IL-4 and/or an IL-5 antagonist, for example an IL-4 antibody or epitope binding domain, and/or an IL-5 antibody or epitope binding domain. These may be administered as a mixture of separate molecules which are administered at the same time i.e. co-administered, or are administered within 24 hours of each other, for example within 20 hours, or within 15 hours or within 12 hours, or within 10 hours, or within 8 hours, or within 6 hours, or within 4 hours, or within 2 hours, or within 1 hour, or within 30 minutes of each other.
  • the antagonists are present as one molecule capable of binding to two or more antigens, for example the invention provides an antigen binding protein comprising the IL-13 antibody of the present invention which is capable of binding to IL-13 and which is also capable of binding to IL-4 or which is also capable of binding to IL-5, or which is also capable of binding to IL-4 and IL-5.
  • the antigen binding protein of the present invention may be a multi-specific antibody which comprises at least CDRH3, and optionally one or more of CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3 of the present invention, which is capable of binding to IL-13 and which is also capable of binding to one or more of IL-4 or IL-5.
  • a multi-specific antibody is provided which comprises a CDRH3, or an antigen binding protein as defined herein, and which comprises a further antigen binding site which is capable of binding to IL-4, or IL-5.
  • an antigen binding protein of the present invention is an antibody specific for IL-13 comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as defined herein, linked to one or more epitope-binding domains which have specificity for IL-4 or IL-5, for example a bispecific antigen binding protein which is capable of binding to IL-13 and IL-4, or IL-13 and IL-5, or a trispecific antigen binding protein which is capable of binding to IL-13, IL-4 and IL-5.
  • any of the antigen-binding proteins described herein may be capable of binding two or more antigens simultaneously, for example, as determined by stochiometry analysis by using a suitable assay such as that described in Example 8.
  • the present invention provides an antigen-binding protein comprising the IL-13 antibody of the present invention which is linked to one or more epitope-binding domains, for example an antigen-binding protein comprising the IL-13 antibody of the present invention linked to an epitope-binding domain which is capable of binding to IL-4, or an antigen-binding protein comprising the IL-13 antibody of the present invention linked to an epitope-binding domain which is capable of binding to IL-5, or an antigen-binding protein comprising the IL-13 antibody of the present invention linked to a first epitope-binding domain which is capable of binding to IL-4 and a second epitope-binding domain which is capable of binding to IL-5.
  • the epitope-binding domain may be attached to the c-terminus or the n-terminus of the heavy chain of the IL-13 antibody or the c-terminus or n-terminus of the light chain of the IL-13 antibody.
  • Suitable linkers include amino acid sequences which may be from 1 amino acid to 150 amino acids in length, or from 1 amino acid to 140 amino acids, for example, from 1 amino acid to 130 amino acids, or from 1 to 120 amino acids, or from 1 to 80 amino acids, or from 1 to 50 amino acids, or from 1 to 20 amino acids, or from 1 to 10 amino acids, or from 5 to 18 amino acids.
  • Such sequences may have their own tertiary structure, for example, a linker of the present invention may comprise a single variable domain.
  • the size of a linker in one embodiment is equivalent to a single variable domain.
  • Suitable linkers may be of a size from 1 to 20 angstroms, for example less than 15 angstroms, or less than 10 angstroms, or less than 5 angstroms.
  • At least one of the epitope binding domains is directly attached to the IL-13 antibody with a linker comprising from 1 to 150 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids.
  • Such linkers may be selected from any one of those set out in SEQ ID NO:82-87, 92 to 93. or multiples of such linkers.
  • Linkers of use in the antigen-binding proteins of the present invention may comprise alone or in addition to other linkers, one or more sets of GS residues, for example ‘GSTVAAPS’ (SEQ ID NO: 92) or TVAAPSGS' (SEQ ID NO: 87) or ‘GSTVAAPSGS’ (SEQ ID NO: 93).
  • the linker comprises SEQ ID NO: 83.
  • the epitope binding domain is linked to the IL-13 antibody by the linker ‘(PAS) n (GS) m ’. In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘(GGGGS) n (GS) m ’. In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘(TVAAPS) n (GS) m ’. In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘(GS) m (TVAAPSGS) n ’. In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘(PAVPPP) n (GS) m ’.
  • the epitope binding domain is linked to the IL-13 antibody by the linker ‘TVAAPS’ (SEQ ID NO: 83). In another embodiment the epitope binding domain, is linked to the IL-13 antibody by the linker TVAAPSGS' (SEQ ID NO: 87). In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘GS’. In another embodiment the epitope binding domain is linked to the IL-13 antibody by the linker ‘ASTKGPT’ (SEQ ID NO: 84).
  • Epitope-binding domains of use in the present invention are domains that specifically bind an antigen or epitope independently of a different V region or domain, this may be a domain antibody or may be a non-Immunoglobulin domain, for example a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human ⁇ -crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to
  • this may be an domain antibody or other suitable domains such as a domain selected from the group consisting of CTLA-4, lipocallin, SpA, an Affibody, an avimer, GroEI, transferrin, GroES and fibronectin.
  • this may be selected from an immunoglobulin single variable domain, an Affibody, an ankyrin repeat protein (DARPin) and an adnectin.
  • this may be selected from an Affibody, an ankyrin repeat protein (DARPin) and an adnectin.
  • this may be a domain antibody, for example a domain antibody selected from a human, camelid (nanobody), or shark (NARV) domain antibody.
  • antigen-binding proteins include the IL-13 antibodies of the present invention which have an epitope binding domain which is an IL-4 antagonist attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus.
  • antigen binding protein comprising the heavy chain sequence set out in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106 or SEQ ID NO: 117-138, and the light chain sequence set out in SEQ ID NO: 24, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO
  • the antigen binding constructs of the present invention comprise the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 64 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 66 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 68 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 70 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 72 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO:74 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 76 and the light chain sequence of SEQ ID NO: 24.
  • the antigen binding constructs of the present invention comprise the heavy chain sequence of SEQ ID NO: 94 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 96 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 98 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 100 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 102 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO: 104 and the light chain sequence of SEQ ID NO: 24, or the heavy chain sequence of SEQ ID NO:106 and the light chain sequence of SEQ ID NO: 24.
  • the antigen binding constructs of the present invention comprise the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 64 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 66 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 68 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 70 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 72 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO:74 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 76 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 110, or the heavy chain chain sequence
  • the antigen binding constructs of the present invention comprise the heavy chain sequence of SEQ ID NO: 96 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 98 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 100 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 102 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 104 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 106 and the light chain sequence of SEQ ID NO: 108, or the heavy chain sequence of SEQ ID NO: 96 and the light chain sequence of SEQ ID NO: 110, or the heavy chain sequence of SEQ ID NO: 98 and the light chain sequence of SEQ ID NO: 110, or the heavy chain sequence of SEQ ID NO: 100 and the light chain sequence of SEQ ID NO: 110, or the heavy chain chain
  • the IL-13 antibody heavy chain is selected from those set out in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54. In another embodiment the heavy chain is selected from those set out in SEQ ID NO:88-91, SEQ ID NO:96-106, and SEQ ID NO:117-138. In one such embodiment the heavy chain is selected from SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104 and SEQ ID NO:106.
  • the antigen-binding protein will comprise an anti-IL-13 antibody linked to an epitope binding domain which is a IL-5 antagonist, wherein the anti-IL-13 antibody comprises the CDRH3 selected from those set out in SEQ ID NO: 3-18, for example SEQ ID NO: 15-18 and the light chain sequence of SEQ ID NO: 24.
  • Examples include an antigen binding protein comprising the heavy chain sequence set out in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 and the light chain sequence set out in SEQ ID NO: 24 wherein one or both of the Heavy and Light chain further comprise one or more epitope-binding domains which is capable of antagonising IL-5, for example immunoglobulin single variable domains which are capable of binding to IL-5.
  • the antigen binding protein will comprise the heavy chain sequence set out in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 and the light chain sequence set out in SEQ ID NO: 24 wherein one or both of the Heavy and Light chain further comprise one or more epitope-binding domains which are capable of antagonising IL-4 for example immunoglobulin single variable domains which are capable of binding to IL-4, and one or more epitope-binding domains which are capable of antagonising IL-5, for example immunoglobulin single variable domains which are capable of binding to IL-5.
  • the antigen-binding protein of the present invention comprises at least one epitope binding domain, which is capable of binding human serum albumin.
  • there are at least 3 antigen-binding sites for example there are 4, or 5 or 6 or 8 or 10 antigen-binding sites and the antigen-binding protein is capable of binding at least 3 or 4 or 5 or 6 or 8 or 10 antigens, for example it is capable of binding 3 or 4 or 5 or 6 or 8 or 10 antigens simultaneously.
  • a first epitope binding domain is linked to the protein scaffold and a second epitope binding domain is linked to the first epitope binding domain
  • the protein scaffold is an IgG scaffold
  • a first epitope binding domain may be linked to the c-terminus of the heavy chain of the IgG scaffold, and that epitope binding domain can be linked at its c-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the c-terminus of the light chain of the IgG scaffold, and that first epitope binding domain may be further linked at its c-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the n-terminus of the light chain of the IgG scaffold, and that first epitope binding domain may be further linked at its n-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the n-terminus of the heavy chain of
  • the epitope-binding domain is a domain antibody
  • some domain antibodies may be suited to particular positions within the scaffold.
  • Immunoglobulin single variable domains of use in the present invention can be linked at the C-terminal end of the heavy chain and/or the light chain of the IL-13 mAb. In addition some immunoglobulin single variable domains can be linked to the C-terminal ends of both the heavy chain and the light chain of conventional antibodies.
  • a peptide linker may help the immunoglobulin single variable domain to bind to antigen.
  • the N-terminal end of a dAb is located closely to the CDRs involved in antigen-binding activity.
  • a short peptide linker acts as a spacer between the epitope-binding domain, and the constant domain of the antibody, which may allow the dAb CDRs to more easily reach the antigen, which may therefore bind with high affinity.
  • immunoglobulin single variable domains are linked to the IgG.
  • each immunoglobulin single variable domain When fused at the C-terminal end of the antibody light chain, each immunoglobulin single variable domain is expected to be located in the vicinity of the antibody hinge and the Fc portion. It is likely that such immunoglobulin single variable domains will be located far apart from each other. In conventional antibodies, the angle between Fab fragments and the angle between each Fab fragment and the Fc portion can vary quite significantly. It is likely that—with mAbdAbs—the angle between the Fab fragments will not be widely different, whilst some angular restrictions may be observed with the angle between each Fab fragment and the Fc portion.
  • each immunoglobulin single variable domain is expected to be located in the vicinity of the C H 3 domains of the Fc portion. This is not expected to impact on the Fc binding properties to Fc receptors (e.g. Fc ⁇ RI, II, III an FcRn) as these receptors engage with the C H 2 domains (for the Fc ⁇ RI, II and III class of receptors) or with the hinge between the C H 2 and C H 3 domains (e.g. FcRn receptor).
  • Fc receptors e.g. Fc ⁇ RI, II, III an FcRn
  • both immunoglobulin single variable domains are expected to be spatially close to each other and provided that flexibility is provided by provision of appropriate linkers, these immunoglobulin single variable domains may even form homodimeric species, hence propagating the ‘zipped’ quaternary structure of the Fc portion, which may enhance stability of the construct.
  • Such structural considerations can aid in the choice of the most suitable position to link an epitope-binding domain, for example an immunoglobulin single variable domain, on to an antibody.
  • the size of the antigen, its localization (in blood or on cell surface), its quaternary structure (monomeric or multimeric) can vary.
  • Conventional antibodies are naturally designed to function as adaptor constructs due to the presence of the hinge region, wherein the orientation of the two antigen-binding sites at the tip of the Fab fragments can vary widely and hence adapt to the molecular feature of the antigen and its surroundings.
  • immunoglobulin single variable domains linked to an antibody with no hinge region may have less structural flexibility either directly or indirectly.
  • Ig domains such as Bence-Jones proteins (which are dimers of immunoglobulin light chains (Epp et al (1975) Biochemistry 14 p 4943-4952; Huan et al (1994) Biochemistry 33 p 14848-14857; Huang et al (1997) Mol immunol 34 p 1291-1301) and amyloid fibers (James et al. (2007) J Mol. Biol. 367:603-8).
  • dAbs that tend to dimerise in solution to the C-terminal end of the Fc portion in preference to the C-terminal end of the light chain as linking to the C-terminal end of the Fc will allow those dAbs to dimerise in the context of the antigen-binding protein of the invention.
  • the antigen-binding proteins of the present invention may comprise antigen-binding sites specific for a single antigen, or may have antigen-binding sites specific for two or more antigens, or for two or more epitopes on a single antigen, or there may be antigen-binding sites each of which is specific for a different epitope on the same or different antigens.
  • the antigen binding proteins of the invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or functional fragment or equivalent thereof.
  • An antigen binding protein of the invention may therefore comprise the VH regions of the invention formatted into a full length antibody, a (Fab′) 2 fragment, a Fab fragment, or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain.
  • the antibody may be an IgG1, IgG2, IgG3, or IgG4; or IgM; IgA, IgE or IgD or a modified variant thereof.
  • the constant domain of the antibody heavy chain may be selected accordingly.
  • the light chain constant domain may be a kappa or lambda constant domain.
  • the antigen binding protein may comprise modifications of all classes e.g. IgG dimers, Fc mutants that no longer bind Fc receptors or mediate C1q binding.
  • the antigen binding protein may also be a chimeric antibody of the type described in WO86/01533 which comprises an antigen binding region and a non-immunoglobulin region.
  • the constant region is selected according to any functionality required.
  • An IgG1 may demonstrate lytic ability through binding to complement and/or will mediate ADCC (antibody dependent cell cytotoxicity).
  • An IgG4 can be used if a non-cytotoxic blocking antibody is required.
  • IgG4 antibodies can demonstrate instability in production and therefore an alternative is to modify the generally more stable IgG1. Suggested modifications are described in EP0307434, for example mutations at positions 235 and 237.
  • the invention therefore provides a lytic or a non-lytic form of an antigen binding protein, for example an antibody according to the invention.
  • the antibody of the invention is a full length (e.g. H2L2 tetramer) lytic or non-lytic IgG1 antibody having any of the heavy chain variable regions described herein.
  • the invention provides polynucleotides encoding the light and heavy chain variable regions as described herein.
  • the antigen-binding site binds to antigen with a Kd of at least about 1 mM, for example a Kd of at least about 10 nM, at least about 1 nM, at least about 500 ⁇ M, at least about 200 ⁇ M, at least about 100 ⁇ M, or at least about 50 ⁇ M to each antigen as measured by BiacoreTM.
  • the antigen-binding site binds to antigen with a Kd of at least about 1 mM, for example a Kd of at least about 10 nM, at least about 1 nM, at least about 500 ⁇ M, at least about 200 ⁇ M, at least about 100 ⁇ M, or at least about 50 ⁇ M to each antigen as measured by BiacoreTM.
  • neutralises and grammatical variations thereof as used throughout the present specification in relation to antigen binding proteins of the invention means that a biological activity of IL-13 is reduced, either totally or partially, in the presence of the antigen binding proteins of the present invention in comparison to the activity of IL-13 in the absence of such antigen binding proteins.
  • Neutralisation may be due to but not limited to one or more of blocking ligand binding, preventing the ligand activating the receptor, down regulating the IL-13 receptor or affecting effector functionality.
  • Levels of neutralisation can be measured in several ways, for example by use of the assays as set out in the examples below, for example in a TF1 assay which may be carried out for example as described in Example 4.
  • the neutralisation of IL-13, IL-4 or both of these cytokines in this assay is measured by assessing the inhibition of TF1 cell proliferation in the presence of neutralising antigen binding protein.
  • assessing neutralisation for example, by assessing the decreased binding between the IL-13 and its receptor in the presence of neutralising antigen binding protein are known in the art, and include, for example, Biacore assays.
  • antigen binding proteins which have at least substantially equivalent neutralising activity to the antibodies exemplified herein, for example antigen binding proteins which retain the neutralising activity of A1Y100BAlaL1, A1Y100BIleL1, A1Y100BTrpL1 or A1Y100BValL1 in a TF1 cell proliferation assay which can be carried out as set out in Example 4.
  • the antigen binding proteins for example antibodies of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antigen binding protein of the invention.
  • An expression vector or recombinant plasmid is produced by placing these coding sequences for the antigen binding protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
  • Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies.
  • a second expression vector can be produced having a DNA sequence which encodes a complementary antigen binding protein light or heavy chain.
  • this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed.
  • the heavy and light chain coding sequences for the antigen binding protein may reside on a single vector.
  • a selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains.
  • the transfected cell is then cultured by conventional techniques to produce the engineered antigen binding protein of the invention.
  • the antigen binding protein which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antigen binding proteins.
  • Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors may be used.
  • One vector, pUC19 is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).
  • any vector which is capable of replicating readily has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning.
  • the selection of the cloning vector is not a limiting factor in this invention.
  • the expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR).
  • Other vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro).
  • BGH bovine growth hormone
  • betaglopro betaglobin promoter sequence
  • replicons e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • selection genes e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
  • the present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antigen binding proteins of the present invention.
  • Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. coli may be used for replication of the cloning vectors and other steps in the construction of antigen binding proteins of this invention.
  • Suitable host cells or cell lines for the expression of the antigen binding proteins of the invention include mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell.
  • mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell.
  • Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.
  • other eukaryotic cell lines may be employed.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art
  • Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs or other embodiments of the present invention (see, e.g., Plückthun, A., Immunol. Rev., 130:151-188 (1992)).
  • any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability.
  • the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule may express in the bacterial host and then be subsequently re-folded.
  • various strains of E. coli used for expression are well-known as host cells in the field of biotechnology.
  • Various strains of B. subtilis, Streptomyces , other bacilli and the like may also be employed in this method.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.
  • the general methods by which the vectors may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding protein of the invention from such host cell may all be conventional techniques.
  • the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension.
  • the antigen binding proteins of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention. For example, preparation of altered antibodies are described in WO 99/58679 and WO 96/16990.
  • Yet another method of expression of the antigen binding proteins may utilize expression in a transgenic animal, such as described in U.S. Pat. No. 4,873,316. This relates to an expression system using the animal's casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.
  • a method of producing an antibody of the invention comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and/or heavy chain of the antibody of the invention and recovering the antibody thereby produced.
  • an anti-IL-13 antibody of the present invention which binds to and neutralises the activity of human IL-13 which method comprises the steps of;
  • the antibody is then examined for in vitro activity by use of an appropriate assay.
  • an appropriate assay Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the antibody to IL-13. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antibody in the body despite the usual clearance mechanisms.
  • the dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient. It is envisaged that repeated dosing (e.g. once a week or once every two weeks) over an extended time period (e.g. four to six months) may be required to achieve maximal therapeutic efficacy.
  • the mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host.
  • the antigen binding proteins, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.), intravenously (i.v.), or intranasally.
  • Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding protein of the invention as an active ingredient in a pharmaceutically acceptable carrier.
  • the prophylactic agent of the invention is an aqueous suspension or solution containing the antigen binding proteinin a form ready for injection.
  • the suspension or solution is buffered at physiological pH
  • the compositions for parenteral administration will comprise a solution of the antigen binding protein of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier.
  • the carrier is an aqueous carrier.
  • a variety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the antigen binding protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as about 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain about 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or about 5 mg to about 25 mg, of an antigen binding protein, for example an antibody of the invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 or 5 mg to about 25 mg of an antigen binding protein of the invention per ml of Ringer's solution.
  • parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.
  • intravenously administrable antigen binding protein formulations of the invention see Lasmar U and Parkins D “The formulation of Biopharmaceutical products”, Pharma. Sci. Tech. today, page 129-137, Vol. 3 (3 rd April 2000); Wang, W “Instability, stabilisation and formulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999) 129-188; Stability of Protein Pharmaceuticals Part A and B ed Ahern T. J., Manning M.
  • the therapeutic agent of the invention when in a pharmaceutical preparation, is present in unit dose forms.
  • the appropriate therapeutically effective dose will be determined readily by those of skill in the art. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of about 0.1 to about 20 mg/kg, for example about 1 to about 20 mg/kg, for example about 10 to about 20 mg/kg or for example about 1 to about 15 mg/kg, for example about 10 to about 15 mg/kg.
  • suitable doses may be within the range of about 0.1 to about 1000 mg, for example about 0.1 to about 500 mg, for example about 500 mg, for example about 0.1 to about 100 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or about 0.1 to about 40 mg, or for example about 1 to about 100 mg, or about 1 to about 50 mg, of an antigen binding protein of this invention, which may be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.
  • the antigen binding proteins described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antigen binding protein of the present invention or a functional fragment thereof and a pharmaceutically acceptable carrier for treatment or prophylaxis of atopic diseases/disorders and chronic inflammatory diseases/disorders, for example, asthma, such as allergic asthma, particularly severe asthma (that is asthma that is unresponsive to current treatment, including systemically administered corticosteroids; see Busse W W et al, J. Allergy Clin.
  • asthma defined as the asthmatic phenotype characterised by failure to achieve control despite maximally recommended doses of prescribed inhaled steroids, see Barnes P J (1998), Eur Respir J 12:1208-1218
  • brittle defined asthma (defines a subgroup of patients with severe, unstable asthma who maintain a wide peak expiratory flow (PEF) variability despite high doses of inhaled steroids, see Ayres J G et al (1998) Thorax 58:315-321), nocturnal asthma, premenstrual asthma, steroid resistant asthma (see Woodcock A J (1993) Eur Respir J 6:743-747), steroid dependent asthma (defined as asthma that can be controlled only with high doses of oral steroids), aspirin induced asthma, adult-onset asthma, paediatric asthma.
  • Antibodies of the invention may be used to prevent, reduce the frequency of, or mitigate the effects of acute, asthmatic episodes (status asthmaticus). Antibodies of the invention may also be used to reduce the dosing required (either in terms of amount administered or frequency of dosing) of other medicaments used in the treatment of asthma. For example, antibodies of the invention may be used to reduce the dosing required for steroid treatment of asthma such as corticosteroid treatment (“steroid sparing”).
  • diseases or disorders that may be treated with antibodies of the invention include atopic dermatitis, allergic rhinitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, and diseases of cell cycle regulation, e.g. Hodgkins disease, B cell chronic lymphocytic leukaemia.
  • the disorder is severe asthma.
  • the disorder is a fibrotic disorder such as IPF.
  • the invention provides a pharmaceutical composition comprising an antigen binding protein of the present invention and a pharmaceutically acceptable carrier for treating atopic diseases/disorders and chronic inflammatory diseases/disorders, for example, asthma, such as allergic asthma, particularly severe asthma (that is asthma that is unresponsive to current treatment, including systemically administered corticosteroids; see Busse W W et al, J. Allergy Clin.
  • asthma such as allergic asthma, particularly severe asthma (that is asthma that is unresponsive to current treatment, including systemically administered corticosteroids; see Busse W W et al, J. Allergy Clin.
  • asthma defined as the asthmatic phenotype characterised by failure to achieve control despite maximally recommended doses of prescribed inhaled steroids, see Barnes P J (1998), Eur Respir J 12:1208-1218
  • brittle defined asthma (defines a subgroup of patients with severe, unstable asthma who maintain a wide peak expiratory flow (PEF) variability despite high doses of inhaled steroids, see Ayres J G et al (1998) Thorax 58:315-321), nocturnal asthma, premenstrual asthma, steroid resistant asthma (see Woodcock A J (1993) Eur Respir J 6:743-747), steroid dependent asthma (defined as asthma that can be controlled only with high doses of oral steroids), aspirin induced asthma, adult-onset asthma, paediatric asthma.
  • Antibodies of the invention may be used to prevent, reduce the frequency of, or mitigate the effects of acute, asthmatic episodes (status asthmaticus). Antibodies of the invention may also be used to reduce the dosing required (either in terms of amount administered or frequency of dosing) of other medicaments used in the treatment of asthma. For example, antibodies of the invention may be used to reduce the dosing required for steroid treatment of asthma such as corticosteroid treatment (“steroid sparing”).
  • diseases or disorders that may be treated with antibodies of the invention include atopic dermatitis, allergic rhinitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, and diseases of cell cycle regulation, e.g. Hodgkins disease, B cell chronic lymphocytic leukaemia.
  • the disorder is severe asthma.
  • the disorder is a fibrotic disorder such as IPF.
  • sequences described herein include sequences which are substantially identical, for example sequences which are at least 90% identical, for example which are at least 91%, or at least 92%, or at least 93%, or at least 94% or at least 95%, or at least 96%, or at least 97% or at least 98%, or at least 99% identical to the sequences described herein.
  • nucleic acids For nucleic acids, the term “substantial identity” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to about 95%, or at least about 98% to about 99.5% of the nucleotides. Alternatively, substantial identity exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • nucleotide and amino acid sequences For nucleotide and amino acid sequences, the term “identical” indicates the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions. Alternatively, substantial identity exists when the DNA segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 25, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
  • Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 25 by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO: 23, or:
  • nn is the number of nucleotide alterations
  • xn is the total number of nucleotides in SEQ ID NO: 25
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn.
  • Alterations of the polynucleotide sequence of SEQ ID NO: 25 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polypeptide sequence of the present invention may be identical to the reference sequence encoded by SEQ ID NO: 24, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 24 by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 24, or:
  • na is the number of amino acid alterations
  • xa is the total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 24, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
  • Original murine mAbs were produced by immunisation of mice with recombinant human IL-13. Spleens from responder animals were harvested and fused to myeloma cells to generate hybridomas. The hybridoma supernatant material was screened for binding. Hybridomas of interest were monocloned using standard techniques.
  • the resulting murine antibody (6A1) comprises the variable regions shown in SEQ ID NO:58 and SEQ ID NO:59. Further details of this murine antibody and a humanised version of this antibody A1L1 (SEQ ID NO: 22 and 24) are described in WO2006/003407 which is herein incorporated by reference.
  • the anti-IL-13 mAb antibody A1L1 was used in several of the following examples as a comparator antibody.
  • a number of variants of the humanised antibody comprising the heavy chain set out in SEQ ID NO: 22 were produced. These all differed in the CDRH3 region of the antibody (SEQ ID NO: 3).
  • the base DNA expression constructs for the antibodies of the present invention were prepared de novo by build-up of overlapping oligonucleotides including restriction sites for cloning into Rld and Rln mammalian expression vectors as well as a human signal sequence.
  • Hind III and Spe I restriction sites were introduced to frame the V H domain containing the signal sequence (SEQ ID NO:56) for cloning into Rld containing the human ⁇ 1 constant region.
  • Hind III and BsiWI restriction sites were introduced to frame the V H domain containing the signal sequence (SEQ ID NO: 56) for cloning into Rln containing the human kappa constant region.
  • Alternative constructs were produced using pTT vectors which also included human constant regions. Where appropriate, site-directed mutagenesis (SDM) was used to generate different humanised constructs.
  • SDM site-directed mutagenesis
  • pTT plasmids encoding the heavy and light chains respectively were transiently co-transfected into HEK 293 6E cells and expressed at small scale to produce antibody. Antibodies were assessed directly from the tissue culture supernatant. Other antibodies were purified using immobilised Protein A columns and quantified by reading absorbance at 280 nm and where indicated, the purified antibody material was assessed in the assays described in the examples set out below.
  • A1Y100BTrpL1 relates to the mAb generated by co-transfection and expression of the noted first and second plasmid, for example ‘A1Y100BTrpL1’ relates to a mAb generated by co-transfection of a plasmid containing the A1Y100BTrp sequence and a plasmid containing the L1 sequence in a suitable cell line.
  • the initial screen of CDRH3 mutants was carried out on the ProteOn XPR36 (Biorad).
  • the method was as follows, antihuman IgG (Biacore BR-1008-39) was immobilised on a GLM chip by primary amine coupling, CDRH3 mutant antibodies were then captured on this surface and IL13 passed over at 256, 64, 16, 4, 1 nM with a OnM injection (i.e. buffer alone) used to double reference. 3M MgCl 2 was used to regenerate the capture surface, removing the bound CDRH3 mutant antibodies ready for another cycle of capture and analyte injection. The data was fitted to the 1:1 model using the software inherent to the machine. All work was carried out using antibodies directly from tissue culture supernatants except for the parental antibody which was purified material.
  • the screen identified several antibodies that appeared to have better kinetic profile than the parental molecule, these same samples were then analysed on the Biacore T100 to confirm the results, using a similar method, in that the same antihuman IgG capture antibody was immobilised on a CM5 chip by primary amine coupling, IL13 was passed over the surface at 256, 64, 16, 4, 1 and 0.25 nM with a OnM used for double referencing, regeneration was with 3M MgCl 2 and the data was fitted to the 1:1 model inherent to the T100. Table 1 details the overall affinities (equilibrium dissociation constant K D ) for the selected constructs from the ProteOn screen and the T100 run
  • A1Y100B VaIL1 was purified and run along side the other purified A1Y100BL1 mutants that also improved affinity using the Biacore T100 machine using the method described earlier.
  • Table 3 shows the data obtained from this experiment. This experiment was in good agreement with the data in Table 2 and confirmed the improvement of affinity for the Y100B mutations.
  • TF-1 cells proliferate in response to a number of different cytokines including human IL-13.
  • the proliferative response of these cells for IL-13 can therefore be used to measure the bioactivity of IL-13 and subsequently an assay has been developed to determine the IL-13 neutralisation potency (inhibition of IL-13 bioactivity) of mAbs.
  • the assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. 14 ng/ml recombinant E. Coli -expressed human IL-13 was pre-incubated with various dilutions of mAbs (usually from 93.4 nM titrated in 3-fold dilutions to 0.014 nM) for 1 hour at 37C. An antibody of irrelevant specificity was similarly titrated as a negative control. These samples were then added to 50 ⁇ l of TF-1 cells (at a concentration of 2 ⁇ 10 5 cells per ml) in a sterile 96-well tissue culture plate.
  • the final 1000 assay volume contained various dilutions of mAbs (at a final concentration of 46.7 nM titrated in 3-fold dilutions to 0.007 nM), recombinant E. Coli -expressed human IL-13 (at a final concentration of 7 ng/ml) and TF-1 cells (at a final concentration of 1 ⁇ 10 5 cells per ml).
  • the assay plate was incubated at 37C for approximately 3 days in a humidified CO 2 incubator.
  • the amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions.
  • the absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.
  • the capacity of the mAbs to neutralise recombinant E. Coli -expressed human IL-13 bioactivity was expressed as that concentration of the mAb required to neutralise the bioactivity of the defined amount of human IL-13 (7 ng/ml) by 50% ( ⁇ ND 50 ).
  • the ND 50 data provided herein (Table 4) were calculated using Robosage in Microsoft Excel. Graphical representation of the data can be seen in FIG. 1 .
  • genes encoding each of the sequences for the variable heavy regions of the CDRH3 variants of the A1 antibodies were transferred from existing constructs to an expression vector containing the hIgG1 constant region fused to an anti-human IL-4 domain antibody (DOM9-112-210) via a TVAAPS or ASTKGPS linker at the c-terminus of the hlgG1 constant region. Details of the heavy chains constructed are listed in Table 5.
  • BPC1624, BPC1625, BPC1626 and BPC1627 were expressed in HEK293 cells. Briefly, 250 ml of HEK293 cells at 1.5 ⁇ 10 6 cells/ml were co-transfected with heavy and light chain expression plasmids previously incubated with 293fectin reagent (Invitrogen # 51-0031). These were placed in a shaking incubator at 37° C., 5% CO 2 , and 95% relative humidity. After 24 hours Tryptone feeding media was added and the cells grown for a further 5 days. Supernatant was harvested by centrifugation and filter sterilised. The expressed molecules were purified by affinity chromatography using immobilised Protein A columns and the concentration was determined by measuring the absorbance at 280 nm. The level of aggregated protein in the purified samples was determined by size exclusion chromatography. The yield of purified protein and levels of aggregation are shown in Table 5b.
  • mAb-dAbs comprising the CDRH3 variant anti-IL-13 mAb were tested for neutralisation of E. Coli -expressed recombinant human IL-13 in a TF-1 cell proliferation bioassay
  • the assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 20 ng/ml recombinant E. Coli -expressed human IL-13 was pre-incubated with various dilutions of mAbdAbs (usually from 50 nM titrated in 3-fold dilutions to 0.02 nM) (those mAbdAbs made in HEK cells and purified as described in example 5) in a total volume of 500 for 1 hour at 37° C. An antibody of irrelevant specificity was similarly titrated as a negative control (data not shown).
  • TF-1 cells at a concentration of 2 ⁇ 10 5 cells per ml
  • the final 1000 assay volume contained various dilutions of mAbdAbs (at a final concentration of 25 nM titrated in 3-fold dilutions to 0.01 nM), recombinant E. Coli -expressed human IL-13 (at a final concentration of 10 ng/ml) and TF-1 cells (at a final concentration of 1 ⁇ 10 5 cells per ml).
  • the assay plate was incubated at 37° C. for approximately 3 days in a humidified CO 2 incubator.
  • the amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturer's instructions.
  • the absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.
  • the capacity of the mAbdAbs to neutralise human IL-13 bioactivity was expressed as that concentration of the mAb-dAb required to neutralise the bioactivity of the defined amount of human IL-13 (10 ng/ml) by 50% ( ⁇ ND 50 ). The lower the concentration of the mAbdAb required, the more potent the neutralisation capacity.
  • the ND 50 data provided herein (Table 6) were calculated using Graph Pad Prism. These data are represented graphically in FIG. 2 .
  • Molecules BPC 1624 to 1631 as shown in Table 5 were also expressed in CHOE1a cells.
  • DNA vectors encoding the heavy and light chains were co-electroporated into suspension CHO cells.
  • Cells were passaged in shake flasks in MR1 basal selective medium at 37° C., 5% CO 2 , 130 rpm until cell viability and cell counts improved.
  • CHO cells were then inoculated into MR1 basal x2 selective medium and incubated for 8 to 12 days at 34° C., 5% CO 2 , 130 rpm. The cells were pelleted by centrifugation and the supernatant sterile filtered.
  • Expressed material was purified by affinity chromatography using immobilised protein A columns and the yield determined by measurement of absorbance at 280 nm. The level of aggregates was determined by size exclusion chromatography. Aggregates were removed by preparative size exclusion chromatography and the yield re-assessed. Table 7 lists the yields and levels of aggregate obtained from this expression system.
  • This example is prophetic. It provides guidance for carrying out an additional assay in which the antigen binding proteins of the invention can be tested,
  • Anti-human IgG is immobilised onto a CM5 biosensor chip by primary amine coupling. Antigen binding proteins are captured onto this surface after which a single concentration of IL-13 or IL-4 or IL-5 is passed over, this concentration is enough to saturate the binding surface and the binding signal observed reached full R-max. Stoichiometries are then calculated using the given formula:
  • the different antigens are passed over sequentially at the saturating antigen concentration and the stoichometries calculated as above.
  • the work can be carried out on the Biacore 3000, at 25° C. using HBS-EP running buffer.
  • An antibody-ligand binding PK-PD model was developed in order to rank the different monoclonal antibody (mAb) candidates based on binding affinity and predicted potential therapeutic dose in human.
  • the predicted potential therapeutic dose in human was defined for this purpose as the dose providing 90% inhibition of the target IL-13 in the lung (site of action) at steady-state following monthly intravenous administration of the mAbs for 1 h.
  • the molecular weight of each molecule was assumed to be the same and equal to the standard molecular weight of a mAb i.e. 150 kDa.
  • the human pharmacokinetics of the A1L1 antibody was inferred to all the candidates.
  • the same antibody-ligand binding PK-PD model is used for each mAb as well as the same assumptions regarding the target concentration, the target turnover, the target tissue:plasma ratio and the mAb tissue penetration.
  • the ranking provided by the model is therefore solely based on the binding affinity of the molecules, the only parameter differing.
  • the potential therapeutic dose in human for the 4 candidates A1Y100BIleL1, A1Y100BValL1, A1Y100BAlaL1 and A1Y100BTrpL1 is predicted to provide a substantial improvement above the predicted potential therapeutic dose in human for A1L1.
  • the anti IL-4 dAb (DOM9-155-154, SEQ ID NO: 80), was investigated for aggregation-prone residues using an aggregation prediction algorithm.
  • the leucine residue, at Kabat position 89 was identified as a key residue for promotion of aggregation.
  • Plasmids encoding the heavy and light chains respectively were transiently co-transfected into HEK 293 6E cells and expressed at small scale to produce antibody molecules. A tryptone feed was added to each cell culture up to 24 hours after transfection and the cells were harvested after 3 days. Antibody molecules were assessed directly from the tissue culture supernatant and quantified using the Gyrolab workstation.
  • Antibodies produced from small scale transient HEK 2936E transfections were quantified from tissue culture supernatants by a quantitative immunoassay using a Gyrolab Bioaffy Workstation (Gyros).
  • Antibody was captured via the Fc region using a biotinylated anti-IgG Affibody molecule (Abcam) immobilised onto streptavidin-coated particles on a compact disc (CD) microlaboratory (Gyros).
  • the Affibody reagent was vortexed briefly and diluted with PBS-Tween 20 (0.01% v/v) to a final working concentration of 0.1 mg/ml.
  • Antibody was then detected by an ALEXA 647 labelled Fab2 anti-human IgG kappa light chain molecule using laser-induced fluorescence.
  • the ALEXA 647 labelled detection reagent was prepared by vortexing briefly and by centrifugation at 13000 rpm for 4 minutes.
  • the labelled Fab2 detection reagent was added to unlabelled Fab2 which were diluted to final concentrations of 75 nM and 1.5 ⁇ M respectively using Rexcip F Detection reagent diluant (Gyros).
  • the antibody quantification range was between 0.244-250 ⁇ g/ml relative to an anti-CD23 monoclonal antibody standard curve.
  • the anti-CD23 (1 mg/ml) standard curve was generated by serial dilution of the antibody with tissue culture media (Freestyle 293 Expression Media, Pluronic F68 and Geneticin, Invitrogen).
  • the antibody molecules were purified using immobilised Protein A columns and quantified by reading absorbance at 280 nm and where indicated, the purified antibody molecule was assessed in the assays described in the examples set out below.
  • 96-well high binding plates were coated with 5 ⁇ g/ml human IL-4 (made at GSK) in NaHCO 3 and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 ⁇ L of blocking solution (1% BSA in TBST buffer) was added in each well and the plates were incubated for at least one hour at room temperature. The purified mAbdAbs were successively diluted across the plates in blocking solution. After one hour incubation, the plates were washed three times.
  • Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma A7164) was diluted in blocking solution to 1 ⁇ g/mL and 50 ⁇ L was added to each well. The plates were incubated for one hour. After another three washing steps, 50 ⁇ l of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped after about 5 minutes by addition of 25 ⁇ L of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.
  • OPD o-phenylenediamine dihydrochloride
  • the experiment was carried out using mAbdAbs directly from tissue culture supernatants except for the positive control (anti-IL-4 mAb) and the anti-IL13 negative control mAb, which were purified material. These data are shown in FIG. 17 .
  • the result of the ELISA shows that most of these transiently expressed anti-IL13 mAb-anti-IL4 dAbs bound IL-4, but some variation in IL-4 binding activity was observed.
  • the purified positive control anti-IL-4 mAb also showed binding to IL-4, whereas the purified negative control mAb showed no binding to human IL-4.
  • Plasmids encoding heavy chains consisting of an anti-IL-13 mAb and an anti-IL-4 dAb were used as base constructs to generate alternative plasmid constructs.
  • a two step cloning strategy was required.
  • step 1 the DNA sequence encoding the VH of the anti-IL13 mAb component of the H chain was replaced with the DNA sequence encoding the VH of another humanized anti-IL13 antibody (SEQ ID NO:54) by restriction cloning using HindIII and SpeI.
  • step 2 the codon encoding the leucine at Kabat position 89 in the anti-IL4 dAb (DOM9-155-154, SEQ ID NO: 80) component of the mAbdAb was mutated by site directed mutagenesis to glutamine. All of the resulting heavy chain DNA sequences generated are given in SEQ ID NOs: 96, 98 and 100. Table 9 provides a list of the molecules constructed and expressed.
  • Heavy and light chain expression plasmids encoding BPC1085, BPC1086 and BPC1087 mAbdAbs were co-transfected into HEK 2936E cells using 293fectin (Invitrogen, 12347019). A tryptone feed was added to each of the cell cultures after 24 hours and the cells were harvested after 72 hours. The antibodies were purified using a Protein A column before being tested in binding assays.
  • BPC1085, BPC1086 and BPC1087 mAbdAbs were purified using Protein A affinity.
  • 1 ml Protein A columns were used (GE Healthcare) on the AKTA Xpress system, columns were equilibrated in PBS (Gibco/Invitrogen) and the antibodies eluted using Pierce IgG elute.
  • Eluted fractions were neutralised using 1M Tris (Hydroxymethyl) Aminomethane buffer (in general 5-10% v/v).
  • Eluted antibody fractions were pooled and analysed for aggregation by size exclusion chromatography and quantified by reading at OD 280 nm using a spectrophotometer.
  • BPC2222, 2223, 2230 and 2231 mAbdAbs were purified using Protein A affinity. 1 ml Protein A columns were used (GE Healthcare) on the AKTA Xpress system, columns were equilibrated in PBS (Gibco/Invitrogen) and the antibodies eluted using Pierce IgG elute. Eluted fractions were neutralised using 1M Tris (Hydroxymethyl) Aminomethane buffer (in general 5-10% v/v). Eluted antibody fractions were pooled and analysed for aggregation by size exclusion chromatography and quantified by reading at OD 280 nm using a spectrophotometer.
  • BPC2222, 2223, 2230 and 2231 showed aggregation of between 30-40%, with the aggregated material eluting before 10 minutes.
  • BPC1085, BPC1086 and BPC1087 mAbdAbs were tested for binding to IL-4 in a direct binding ELISA according to the method described in Example 10.3.
  • BPC1085, BPC1086 and BPC1087 mAbdAbs were tested for neutralization of human IL-4 in a TF-1 cell bioassay.
  • TF-1 cells proliferate in response to a number of different cytokines including human IL-4.
  • the proliferative response of these cells for IL-4 can therefore be used to measure the bioactivity of IL-4 and subsequently an assay has been developed to determine the IL-4 neutralisation potency (inhibition of IL-4 bioactivity) of mAbdAbs.
  • the assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in duplicate. Approximately 2.2 ng/ml recombinant E. Coli -expressed human IL-4 was pre-incubated with various dilutions of mAbdAbs (usually from 560 nM titrated in 3-fold dilutions to 0.009 nM) in a total volume of 1200 for 1 hour at 37° C. An antibody of irrelevant specificity was similarly titrated as a negative control (anti-IL13 mAb). 500 of these samples were then added to 500 of TF-1 cells (at a concentration of 2 ⁇ 10 5 cells per ml) in a sterile 96-well tissue culture plate.
  • the final 1000 assay volume contained various dilutions of mAbdAbs (at a final concentration of 270 nM titrated in 3-fold dilutions to 0.005 nM), recombinant E. Coli -expressed human IL-4 (at a final concentration of 1.1 ng/ml) and TF-1 cells (at a final concentration of 1 ⁇ 10 5 cells per ml).
  • the assay plate was incubated at 37° C. for approximately 4 days in a humidified CO 2 incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions.
  • the capacity of the mAbdAbs to neutralise recombinant E. Coli -expressed human IL-4 bioactivity was expressed as that concentration of the mAb-dAb required to neutralise the bioactivity of the defined amount of human IL-4 (1.1 ng/ml) by 50% ( ⁇ ND 50 ). The lower the concentration of the mAbdAb required, the more potent the neutralisation capacity.
  • the ND 50 data provided herein (Table 11) were calculated using the Robosage function in Excel. These data are represented graphically in FIG. 11 .
  • An anti-IL-4 mAb and DOM9-155-154 (SEQ ID NO: 80) were included as positive controls for neutralization of human and cynomolgus IL-4 in the TF-1 cell bioassays. Additionally, a dAb with specificity for an irrelevant antigen (dummy dAb) was also included as a negative control for neutralization of human or cynomolgus IL-4 in the TF-1 cell bioassays.
  • ND 50 values were calculated from the dataset.
  • the ND 50 value is the concentration of the mAbdAb or mAb or dAb, which is able to neutralise the bioactivity of IL-4 by 50%.
  • BPC1085, BPC1086 and BPC1087 mAbdAbs were tested for neutralization of human IL-13 in a TF-1 cell bioassay as described below.
  • TF-1 cells proliferate in response to a number of different cytokines including human IL-13.
  • the proliferative response of these cells for IL-13 can therefore be used to measure the bioactivity of IL-13 and subsequently an assay has been developed to determine the IL-13 neutralisation potency (inhibition of IL-13 bioactivity) of mAbdAbs.
  • the assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in duplicate. Approximately 14 ng/ml recombinant E.
  • Coli -expressed human IL-13 was pre-incubated with various dilutions of mAbdAbs (usually from 560 nM titrated in 3-fold dilutions to 0.009 nM) in a total volume of 1200 for 1 hour at 37° C.
  • An antibody and dAb of irrelevant specificity was similarly titrated as negative controls (anti-IL-4 mAb and DOM9-155-154 respectively).
  • 500 of these samples were then added to 500 of TF-1 cells (at a concentration of 2 ⁇ 10 5 cells per ml) in a sterile 96-well tissue culture plate.
  • the final 1000 assay volume contained various dilutions of mAbdAbs (at a final concentration of 270 nM titrated in 3-fold dilutions to 0.005 nM), recombinant E. Coli -expressed human IL-13 (at a final concentration of 7 ng/ml) and TF-1 cells (at a final concentration of 1 ⁇ 10 5 cells per ml).
  • the assay plate was incubated at 37° C. for approximately 4 days in a humidified CO 2 incubator.
  • the amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturer's instructions.
  • the capacity of the mAbdAbs to neutralise recombinant E. Coli -expressed human IL-13 bioactivity was expressed as that concentration of the mAb-dAb required to neutralise the bioactivity of the defined amount of human IL-13 (7 ng/ml) by 50% ( ⁇ ND 50 ). The lower the concentration of the mAbdAb required, the more potent the neutralisation capacity.
  • the ND 50 data provided herein (Table 12) were calculated using the Robosage function in Excel. These data are represented graphically in FIG. 12 .
  • An anti IL-13 mAb (SEQ ID NO:22 & 24) was included as a positive control for neutralization of human IL-13 in the TF-1 cell bioassays. Additionally, an anti-IL-4 mAb was also included as a negative control.
  • FIG. 12 shows the result of the TF-1 cell neutralization assay.
  • ND 50 values were calculated from the dataset.
  • the ND 50 value is the concentration of the mAbdAb or mAb or dAb, which is able to neutralise the bioactivity of IL-13 by 50%.
  • the mean ND 50 value and the number of times tested are shown in Table 12.
  • the light chain CDRs of the murine antibody 6A1 (The light chain of which is set out in SEQ ID NO:59) were re-grafted onto new frameworks in order to improve the expression of some anti-IL-13 mAb-anti-IL-4 dAb molecules (BPC1085).
  • Codon optimised light chain variable region sequences (summarised in Table 13) were constructed de novo using a PCR-based strategy and overlapping oligonucleotides.
  • PCR primers were designed to incorporate the signal sequence (SEQ ID NO: 56) and to include HindIII and BsiWI restriction sites designed to frame the V L domain and allow cloning into pTT and Rln mammalian expression vectors containing the human kappa C region.
  • Table 13 summarises the re-humanised light chains that have been constructed.
  • Plasmids encoding the A1Y100BVAL1 (SEQ ID NO: 54) heavy chain, the existing light chain (SEQ ID NO: 24) and the re-humanised light chains were transiently co-transfected into HEK 293 6E cells using 293fectin (Invitrogen, 12347019). Plasmids were expressed at small scale (2 ⁇ 0.75 ml culture volumes) to produce antibody. A tryptone feed was added to the cell culture after 24 hours and the cells were harvested after a further 72 hours. Table 14 summarises all of the mAbs which were constructed and expressed.
  • Antibody expression was assessed directly from the tissue culture supernatant, by a quantitative immunoassay using a Gyrolab workstation.
  • Antibodies BPC3208 and BPC3211 containing re-humanised light chains (denoted P0 and P1 respectively), exhibited improved expression yields in comparison to the A1Y100BVAL1 mAb.
  • Q0 and Q1 light chains (BPC3219 and BPC3220) did not improve expression of the anti-IL-13 mAb.
  • Expression data is presented in Table 15.
  • Re-humanised light chains of BPC3208 and BPC3211 exhibited improved expression of the anti-IL-13 mAb, they were examined in the context of an anti-IL-13 mAb-anti IL-4-dAb.
  • Re-humanised light chains P0 and P1 and the 586 (L1) light chain were co-transfected with the 829H-GS(TVAAPSGS) 2 -256 heavy chain (SEQ ID NO: 96, details summarised in Table 16) into HEK 293 6E cells using 293fectin (Invitrogen, 12347019). Plasmids were expressed at the 50 to 500 ml scale to produce antibody molecules. A tryptone feed was added to the cell culture after 24 hours and the cells were harvested after a further 48 hours.
  • Antibodies were purified using immobilised Protein A columns and quantified by reading absorbance at 280 nm and where indicated, the purified antibody molecule was assessed in the assays described in the examples set out below.
  • BPC3214 and BPC3215 were analysed by size exclusion chromatography (SEC) as illustrated in FIGS. 13 and 14 .
  • BPC3214 and BPC3215 were tested for binding to human IL-13 in comparison to BPC1085 (described in Example 10) via a direct binding ELISA.
  • Anti-IL-13 mAb A1Y100BVAL1 and anti-IL-4 mAb were also examined as positive and negative controls respectively.
  • 96-well high binding plates were coated with 500/well of recombinant E. coli - expressed human IL-13 (Batch number: GRITS31061) at 5 ⁇ g/ml and incubated at +4° C. overnight. All subsequent steps were carried out at room temperature. The plates were washed 3 times with phosphate-buffered saline with 0.05% of Tween-20.
  • BPC3214 and BPC3215 were also tested for binding to recombinant E. coli -expressed human IL-4 in a direct binding ELISA.
  • An ELISA was performed as described in example 4, coating 96-well high binding plates with 500/well of recombinant E. coli -expressed human IL-4 at 5 ⁇ g/ml and incubated at +4° C. overnight. These data are shown in FIG. 16 .
  • Direct binding ELISA confirms that BPC3214 and BPC3215 bind to human IL-4.
  • BPC1085 was also examined.
  • BPC3214 exhibits similar IL-4 binding potency to BPC1085.
  • Positive control anti-IL-4 mAb also showed binding to recombinant IL-4 whereas negative control anti-IL-13 mAb A1Y100BVAL1 demonstrated no binding to human IL-4.
  • Binding Affinity of mAbdAbs Comprising the Original IL-13 mAb CDRH3 (BPC2222, BPC2223 & BPC2230-2231) for IL-13 and IL-4 as Assessed by BIAcoreTM Analysis
  • Protein A was immobilised on a Cl chip by primary amine coupling; this surface was used as a capture surface for the antibody molecules to be tested.
  • Recombinant E. coli -expressed human IL13 was used at 256, 64, 16, 4, and 1 nM
  • recombinant E. coli -expressed human IL4 was used at 64, 16, 4, 1 and 0.25 nM
  • OnM i.e. buffer alone
  • Regeneration the Protein A surface was with 100 mM Phosphoric acid.
  • the assay was run at 25° C. using HBS-EP as running buffer. The data was fitted to 1:1 model inherent to the Biacore T100 analysis software.
  • Binding Affinity of mAbdAbs Comprising the original IL-13 mAb CDRH3 (BPC2222, BPC2231) & Variant Anti-IL-13 mAb CDRH3 (BPC1085-1087) for IL-13 and IL-4 as Assessed by BIAcoreTM Analysis
  • Protein A was immobilised on a CM5 chip by primary amine coupling; this surface was used as a capture surface for the antibody molecules to be tested.
  • Recombinant E. coli -expressed human IL13 was used at 256 nM only
  • Recombinant E. coli - expressed human IL4 was used at 64, 16, 4 and 1 nM, with OnM (i.e. buffer alone) used to double reference the binding curves for both IL4 and IL13 binding.
  • Regeneration the Protein A surface was with 50 mM NaOH.
  • the assay was run at 25° C. using HBS-EP as running buffer. The data was fitted to 1:1 model inherent to the Biacore T100 analysis software.
  • Binding Affinity of mAbdAbs comprising a Number of Variant Anti-IL-13 mAb CDRH3 (BPC1085, BPC1090-BPC1095, & BPC1108-BPC1119) for IL-4 as Assessed by BIAcoreTM Analysis
  • Protein A was immobilised on a CM5 chip by primary amine coupling; this surface was used as a capture surface for the antibody molecules to be tested.
  • E. coli - expressed human IL4 was used at 64, 16, 4, 1 and 0.25 nM. All binding curves were double referenced with a OnM injection (i.e. buffer alone).
  • Regeneration of the Protein A surface was with 50 mM NaOH.
  • the assay was run at 25° C. using HBS-EP as running buffer.
  • the data was fitted to 1:1 model inherent to the Biacore T100 analysis software.
  • Protein A was immobilised on a CM5 chip by primary amine coupling; this surface was used as a capture surface for the antibody molecules to be tested.
  • E. coli - expressed human IL13 and cyno IL13 were used 64, 16, 4, 1 and 0.25 nM. All binding curves were double referenced with a OnM injection (i.e. buffer alone).
  • Regeneration of the Protein A surface was with 50 mM NaOH.
  • the assay was run at 25° C. using HBS-EP as running buffer.
  • the data was fitted to 1:1 model inherent to the Biacore T100 analysis software.
  • a number of mAbdAbs were placed in PBS or 50 mM acetate buffer and incubated at 37° C. for up to 14 days. They were then analysed for presence of a visual precipitate, soluble aggregate and adherence to concentration stability.
  • BPC1085, BPC1086, and BPC1087 were investigated in separate studies following IV administration to rats.
  • the PK of BPC1085 was also investigated in cynomologus monkeys following IV administration.
  • PK of all three molecules in rat and BPC1085 in monkey were found to be consistent with that of a standard mAb.
  • FIG. 1 A graph showing the capacity of the mAbdAbs comprising the Y100B variants to neutralise human IL-13 in a TF-1 cell proliferation assay
  • FIG. 2 A graph showing the capacity of the mAbdAbs comprising the Y100B variants to neutralise human IL-13 in a TF-1 cell proliferation assay
  • FIG. 3 SEC trace of BPC2222
  • FIG. 4 SEC trace of BPC2223
  • FIG. 5 SEC trace of BPC2230
  • FIG. 6 SEC trace of BPC2231
  • FIG. 7 SEC trace of BPC1085
  • FIG. 8 SEC trace of BPC1086
  • FIG. 9 SEC trace of BPC1087
  • FIG. 10 A graph showing binding of purified mAbdAbs (BPC1085, BPC1086 and BPC1087) to human IL-4 as determined by ELISA.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.
  • FIG. 11 A graph showing neutralization of human IL-4 by purified mAbdAbs (BPC1085, BPC1086 and BPC1087) to human IL-4 in the TF-1 cell bioassay.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.
  • FIG. 12 A graph showing neutralization of human IL-13 by purified mAbdAbs (BPC1085, BPC1086 and BPC1087) to human IL-13 in the TF-1 cell bioassay.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.
  • FIG. 13 SEC profile for BPC3214.
  • FIG. 14 SEC profile for BPC3215.
  • FIG. 15 A graph showing binding of purified mAbdAbs BPC3214, BPC3215, BPC1085 and control mAbs A1Y100BVAL1 and anti-IL-4 mAb to human IL-13 as determined by ELISA.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.
  • FIG. 16 A graph showing binding of purified mAbdAbs BPC3214, BPC3215, BPC1085 and control mAbs A1Y100BVAL1 and anti-IL-4 mAb to human IL-4 as determined by ELISA.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.
  • FIG. 17 A graph showing binding of transiently expressed mAbdAbs to recombinant E. coli - expressed human IL-4 as determined by ELISA.
  • the IL-4 control mAb is labelled as ‘pascolizumab’.

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US8772459B2 (en) 2009-12-02 2014-07-08 Imaginab, Inc. J591 minibodies and Cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
US8940871B2 (en) 2006-03-20 2015-01-27 The Regents Of The University Of California Engineered anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting
US8940298B2 (en) 2007-09-04 2015-01-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US8951737B2 (en) 1996-05-06 2015-02-10 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US10517969B2 (en) 2009-02-17 2019-12-31 Cornell University Methods and kits for diagnosis of cancer and prediction of therapeutic value
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies

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BR112015024553A2 (pt) * 2013-04-05 2017-10-24 Genentech Inc anticorpo multiespecífico, anticorpo isolado, ácido nucleico isolado, célula hospedeira, método de produção de anticorpo, imunoconjugado, formulação farmacêutica, uso de anticorpo e método de tratamento de indivíduos com distúrbio
US9840553B2 (en) * 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US10035823B2 (en) * 2014-09-29 2018-07-31 The Regents Of The University Of California Compositions for expanding regulatory T cells (TREG), and treating autoimmune and inflammatory diseases and conditions
CR20180162A (es) * 2015-10-02 2018-05-25 Hoffmann La Roche Moléculas biespecíficas de unión a antígeno activadoras de células t
CN118005784A (zh) * 2022-11-08 2024-05-10 上海洛启生物医药技术有限公司 抗il-13长效纳米抗体序列及其应用

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EA200801515A1 (ru) * 2006-01-24 2009-02-27 Домантис Лимитед Лиганды, которые связывают il-4 и/или il-13
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951737B2 (en) 1996-05-06 2015-02-10 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US8940871B2 (en) 2006-03-20 2015-01-27 The Regents Of The University Of California Engineered anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting
US8940298B2 (en) 2007-09-04 2015-01-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US9527919B2 (en) 2007-09-04 2016-12-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US10517969B2 (en) 2009-02-17 2019-12-31 Cornell University Methods and kits for diagnosis of cancer and prediction of therapeutic value
US8772459B2 (en) 2009-12-02 2014-07-08 Imaginab, Inc. J591 minibodies and Cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
US11180570B2 (en) 2009-12-02 2021-11-23 Imaginab, Inc. J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies

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