Patent Grant
7585504
Inflammation is often induced by proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6, platelet-activating factor (PAF), macrophage migration inhibitory factor (MIF), and other compounds. These proinflammatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade.
Inflammatory cytokine cascades contribute to deleterious characteristics, including inflammation and apoptosis, of numerous disorders. Included are chronic and acute disorders characterized by both localized and systemic reactions, including, without limitation, diseases involving the gastrointestinal tract and associated tissues (such as appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis); diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, COPD, adult respiratory distress syndrome, pneumoultramicroscopicsilico-volcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoal and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases and conditions of the skin (such as psoriasis, bums, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and associated tissues (such as vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, restenosis and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Retier's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.
The early proinflammatory cytokines (e.g., TNF, IL-1, etc.) mediate inflammation, and induce the late release of high mobility group protein 1 (HMG1) (also known as HMG-1, HMG1, and HMGB1), a protein that accumulates in serum and mediates delayed lethality and further induction of early proinflammatory cytokines.
HMG1 was first identified as the founding member of a family of DNA-binding proteins termed high mobility group (HMG) that are critical for DNA structure and stability. It was identified nearly 40 years ago as a ubiquitously expressed nuclear protein that binds double-stranded DNA without sequence specificity.
HMG1 binding bends DNA to promote formation and stability of nucleoprotein complexes that facilitates gene transcription of, for example, glucocorticoid receptors and RAG recombinase. The HMG1 molecule has three domains: two DNA binding motifs termed HMG A and HMG B boxes, and an acidic carboxyl terminus. The two HMG boxes are highly conserved 80 amino acid, L-shaped domains. HMG boxes are also expressed in other transcription factors including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.
Recently, it has been found that the HMG A box serves as a competitive inhibitor of HMG proinflammatory action, and the HMG B box has the predominant proinflammatory activity of HMG (See, e.g., US20040005316). HMG1 has been demonstrated to be a long-searched-for nuclear danger signal passively released by necrotic, as opposed to apoptotic cells that will induce inflammation. It has also been shown that HMG1 can be actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of the molecule, which enables translocation from the nucleus to secretory lysosomes and results in the secretion of an acetylated form of HMG1. See, PCT/IB2003/005718. Thus, HMG1 passively released from necrotic cells and HMGB1 actively secreted by inflammatory cells are molecularly different.
Further, HMG1 has been implicated as a cytokine mediator of delayed lethality in endotoxemia. See, e.g., U.S. Pat. Nos. 6,468,533 and 6,448,223. More specifically, it is been demonstrated that bacterial endotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages to release HMG1 as a late response to activation, resulting in elevated serum HMG1 levels that are toxic. Antibodies against HMG1 have been shown to prevent lethality of endotoxin even when antibody administration is delayed until after the early cytokine response. Like other proinflammatory cytokines, HMG1 is a potent activator of monocytes. Intratracheal application of HMG1 causes acute lung injury, and anti-HMG1 antibodies protect against endotoxin-induced lung edema. In addition, serum HMG1 levels are elevated in critically ill patients with sepsis or hemorrhagic shock, and levels are significantly higher in non-survivors as compared to survivors.
Extracellular HMG1 acts as a potent mediator of the inflammatory cascade by signaling via the Receptor for Advanced Glycated End-products (RAGE) and via members of the Toll-like receptor (TLR) family. See, e.g., U.S. patent publication no. US20040053841.
High mobility group protein 2 (HMG2) (also known as HMGB2 and HMG-2) is a close relative of HMG1 that likely originated from gene duplication. It is present in many cell types and shares many if not all of the biochemical properties of HMG1 (Bustin, 1999, Mol. Cell. Biol. 19, 5237-46 and Thomas et al., 2001, Trends Biochem. Sci. 26, 167-74). Although HMG2 is less abundant and has a more restricted distribution than HMG1 in adult mouse tissues, it is relatively abundant in the lymphoid organs, testis and lung where it may also play a role as a mediator of inflammation. Like HMG1, HMG2 is also a significant target antigen of autoantibodies (e.g., perinuclear anti-neutrophil cytoplasmic antibodies) in a number of autoimmune diseases including, systemic rheumatic diseases (Uesugi et al., 1998, J Rheumatol. 25:703-9), ulcerative colitis (Sobajima et al., 1998, Clin Exp Immunol. 111:402-7) and juvenile idiopathic arthritis (Wittemann et al., 1990, Arthritis Rheum. 33:1378-83; Rosenberg et al., 2000, J Rheumatol. 27, 2489-93).
Given the fact that antibodies that bind to HMG1 and polypeptide fragments thereof (e.g., HMG A and HMG B box) have been shown modulate the activity of HMG1 (e.g., proinflammatory activity), and the fact that modulating HMG1 activity in humans may have profound therapeutic uses for many diseases and disorders, there is a need in the art to identify antibodies that specifically bind HMG1 and polypeptide fragments thereof that have high affinity for HMG1 and low immunogenicity. Similarly, molecules that modulate the activity of HMG2 (e.g., antibodies that specifically bind HMG2 and polypeptide fragments) may also be useful therapeutics for a number of diseases and disorders.
The present invention is based in part on the discovery of high affinity antibodies that specifically bind HMG1 (also referred herein as “HMGB1”) and antigenic fragments thereof. The high affinity antibodies of the present invention and pharmaceutical compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of inflammatory diseases and disorders such as sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection. In addition, the high affinity antibodies of the present invention are useful for diagnostic applications.
In one embodiment of the present invention, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG1 polypeptide of a human or other animal, e.g., mammals and invertebrates. In a specific embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising or alternatively consisting of a human HMG1 polypeptide (SEQ ID NO:1 or SEQ ID NO:2). Full-length HMG1 polypeptides of human and other animals are well known in the art (see, e.g., US20040005316; U.S. Pat. Nos. 6,468,533 and 6,448,223).
In another embodiment of the present invention, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG2 (also referred to herein as “HMGB2”) polypeptide of a human or other animal, e.g., mammals and invertebrates. In still another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising or alternatively consisting of a human HMG2 polypeptide (SEQ ID NO:21). Full-length HMG2 polypeptides of human and other animals are well known in the art. See, e.g., Majumdar et al., 1991, Nucleic Acids Res. 19:6643; Shirakawa et al., 1992, J Biol Chem 267:6641-6645.
In another embodiment of the present invention, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) either a HMG1 A box or HMG1 B box of a mammal (or other animals), preferably of a human HMG1 polypeptide. The amino acid sequences of HMG1 A box and HMG1 B box polypeptides of humans and other animals are highly conserved and are well known in the art (see, e.g., US20040005316; U.S. Pat. Nos. 6,468,533; 6,448,223, and US20040053841).
In still another embodiment of the present invention, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) either a HMG2 A box or HMG2 B box of a mammal (or other animals), preferably of a human HMG2 polypeptide. The amino acid sequences of HMG box polypeptides of humans and other animals are highly conserved and are well known in the art. See, e.g., Jantzen et al., 1990, Nature 344:830-6; Kolodrubetz 1990, Nucleic Acids Res. 18:5565; Laudet et al., 1993, Nucleic Acids Res. 21:2493-501 and Thomas et al., 2001, Trends Biochem Sci. 26:167-74.
Also encompassed by the present invention are antibodies which specifically bind to an epitope comprising, or alternatively consisting of (or consisting essentially of) amino acid residues derived from both the A box and B box of HMG1 and/or HMG2. An epitope derived from amino acid residues derived from both the A box B box may be a linear polypeptide derived from the junction of the A and B boxes or may result from the three dimensional confirmation of a polypeptide comprising amino acid residues from both the A and B boxes.
In another embodiment of the present invention, the high affinity antibodies of the present invention specifically bind an antigenic HMGB1 polypeptide fragment comprising, or alternatively consisting of (or consisting essentially of) a polypeptide fragment of human HMGB1 (or other animals).
In another embodiment of the present invention, the high affinity antibodies of the present invention specifically bind an antigenic HMGB2 polypeptide fragment comprising, or alternatively consisting of (or consisting essentially of) a polypeptide fragment of human HMGB2 (or other animals).
It is specifically contemplated that the high affinity antibodies of the present invention may specifically bind acetylated and/or non-acetylated HMG1 and antigenic fragments thereof. It is also specifically contemplated that the high affinity antibodies of the invention may be able to distinguish between the two forms. It is also contemplated that the high affinity antibodies of the present invention may specifically bind acetylated and/or non-acetylated HMG2 and antigenic fragments thereof. It is also specifically contemplated that the high affinity antibodies of the invention may be able to distinguish between the two forms.
In a specific embodiment of the present invention, antibodies that specifically bind HMG1 and antigenic fragments thereof are humanized or human antibodies. In another specific embodiment of the present invention, antibodies that specifically bind HMG2 and antigenic fragments thereof are humanized or human antibodies.
Another embodiment of present invention are antibodies that specifically bind HMG1 and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10−5 M, or of less than 10−6 M, or of less than 10−7 M, or of less than 10−8 M, or of less than 10−9 M, or of less than 10−10 M, or of less than 10−11 M, or of less than 10−12 M, or of less than 10−13 M.
Still another embodiment of present invention are antibodies that specifically bind HMG2 and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10−5 M, or of less than 10−6 M, or of less than 10−7 M, or of less than 10−8 M, or of less than 10−9 M, or of less than 10−10 M, or of less than 10−11 M, or of less than 10−12 M, or of less than 10−13 M.
In another embodiment, the antibody of the invention binds to HMG1 and/or antigenic fragments thereof with a Koff of less than 1×10−3 s−1, or less than 3×10−3 s−1. In other embodiments, the antibody binds to HMG1 and antigenic fragments thereof with a Koff of less than 10−3 s−1, less than 5×10−3 s−1, less than 10−4 s−1, less than 5×10−4 s−1, less than 10−5 s−1, less than 5×10−5 s−1, less than 10−6 s−1, less than 5×10−6 s−1less than 10−7 s−1, less than 5×10−7 s−1, less than 10−8 s−1, less than 5×10−8 s−1, less than 10−9 s−1, less than 5×10−9 s−1, or less than 10−10 s−1.
In yet another embodiment, the antibody of the invention binds to HMG2 and/or antigenic fragments thereof with a Koff of less than 1×10−3 s−1, or less than 3×10−3 s−1. In other embodiments, the antibody binds to HMG2 and antigenic fragments thereof with a Koff of less than 10−3 s−1, less than 5×10−3 s−1, less than 10−4 s−1, less than 5×10−4 s−1, less than 10−5 s−1, less than 5×10−5 s−1, less than 10−6 s−1, less than 5×10−6 s−1, less than 10−7 s−1, less than 5×10−7 s−1, less than 10−8 s−1, less than 5×10−8 s−1, less than 10−9 s−1, less than 5×10−9 s−1, or less than 10−10 s−1.
In another embodiment, the antibody of the invention binds to HMG1 and/or antigenic fragments thereof with an association rate constant or kon rate of at least 105 M−1s−1, at least 5×105 M−1s−1, at least 106 M−1s−1, at least 5×106 M−1s−1, at least 107 M−1s−1, at least 5×107 M−1s−1, or at least 108 M−1s−1, or at least 109 M−1s−1.
In another embodiment, the antibody of the invention binds to HMG2 and/or antigenic fragments thereof with an association rate constant or kon rate of at least 105 M−1s−1, at least 5×105 M−1s−1, at least 106 M−1s−1, at least 5×106 M−1s−1, at least 107 M−1s−1, at least 5×107 M−1s−1, or at least 108 M−1s−1, or at least 109 M−1s−1.
It is contemplated that the high affinity antibodies of the invention may specifically bind to HMG1 and not bind to HMG2 or may specifically bind to HMG2 and not bind to HMG1. It is further contemplated that the high affinity antibodies of the invention may specifically bind to both HMG1 and to HMG2 (e.g., an antibody that specifically recognized an epitope that is present in both HMG1 and HMG2). It is contemplated that the high affinities antibodies of the invention may specifically bind either HMG1 or HMG2 and cross-react with HMG2 or HMG1, respectively. It is further contemplated that the high affinity antibodies of the invention bind HMG1 and HMG2 with either the same or different binding affinities.
The high affinity antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
An additional nonexclusive embodiment of the present invention includes high affinity antibodies of the invention that have certain preferred biochemical characteristics such as a particular isoelectric point (pI) or melting tempeature (Tm).
In one embodiment, the high affinity antibodies of the present invention have a pI ranging from 5.5 to 9.5.
In one embodiment, the high affinity antibodies of the present invention have a Tm ranging from about 65° C. to about 120° C.
Specific embodiments of the invention also include particular antibodies (and fragments thereof) that specifically bind HMG1 with high affinity which have been deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209) and assigned ATCC Deposit Nos. PTA-6142 (Deposited Aug. 4, 2004), PTA-6143 (Deposited Aug. 4, 2004), PTA-6259 (Deposited Oct. 19, 2004) and PTA-6258 (Deposited Oct. 19, 2004) (also referred to herein as “S2”, “S6”, “S16”, and “G4”, respectively). These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Since the strain referred to is being maintained under the terms of the Budapest Treaty, it will be made available to a patent office signatory to the Budapest Treaty.
Other specific embodiments of the invention also include particular antibodies (and fragments thereof) that specifically bind HMG1 with high affinity and comprise at least one of the variable regions disclosed herein (see, e.g.,
Further, any antibody that specifically binds the same epitope (e.g., epitopes within the HMG1 peptides 91-169 or 150-183) as the anti-HMG1 antibodies disclosed herein are included within the invention. In a specific embodiment, an antibody that specifically binds the same epitope as the deposited antibodies are included within the invention. It is specifically contemplated that these antibodies will bind the same epitope as the deposited antibodies with at least equal affinity, or better affinity, or less affinity. Isolated polynucleotides that encode these antibodies (and fragments thereof) are also specific embodiments of the invention.
Isolated polynucleotides that encode any of the high affinity antibodies of the invention are included as embodiments of the invention.
In other embodiments, the invention is directed to compositions, e.g., pharmaceutical compositions, comprising the high affinity antibodies of the present invention in a pharmaceutically acceptable excipient.
In a specific embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to an A box of HMG1 (e.g., an epitope within SEQ ID NOS: 3). In another specific embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to a B box of HMG1 (e.g., an epitope within SEQ ID NOS: 4, 28, 29). In still another specific embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to an epitope derived from both the A box and B box of HMG1 and/or HMG2. It is also contemplated that compositions of the invention may comprise a combination of high affinity antibodies of the invention, for example a combination of antibodies that specifically bind to an A box and antibodies that specifically bind a B box.
Compositions of the invention can comprise the high affinity antibodies of the present invention alone or in combination with other active therapeutic molecules and/or adjuvants such as steroids, other anti-inflammatory molecules, or other antibody therapeutics. More specifically, the compositions of the invention can comprise an antagonist of an early sepsis mediator. The antagonist of an early sepsis mediator is in one embodiment, an antagonist of a cytokine selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6.
In another embodiment of the present invention, the compositions described herein can inhibit a condition mediated or characterized by activation of an inflammatory cytokine cascade including both acute and chronic inflammatory conditions.
In still another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition in an animal CLP sepsis model (e.g., mouse or piglet CLP model).
In yet another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition in an animal arthritis model (e.g., rat AIA, mouse passive or active CIA models).
In still another embodiment of the present invention, the compositions described herein reduce bone loss and/or cartilage damage (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in an animal arthritis model (e.g., rat AIA, mouse passive or active CIA models).
In still another embodiment of the present invention, the compositions described herein reduce bone loss and/or cartilage damage (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in humans.
In another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than Renbrel® (with or without methotrexate) in a rodent arthritis model.
In still another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than Enbrel® (with or without methotrexate) in humans.
In yet another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition in a mouse peritonitis model.
Other contemplated embodiments of the present invention include methods of treating or preventing arthritis, e.g., rheumatoid arthritis, osteoclast-mediated diseases, or other inflammatory diseases of the joints comprising administering an antibody composition described herein.
In another embodiment, the present invention includes methods of treating or preventing arthritis, e.g., rheumatoid arthritis, osteoclast-mediated diseases, or other inflammatory diseases comprising administering any antibody (or antibody composition) that specifically binds HMG1 or antigenic fragment thereof (e.g., HMG B box) irregardless of the binding affinity of the antibody.
In another embodiment, the present invention includes methods of treating or preventing arthritis, e.g., rheumatoid arthritis, osteoclast-mediated diseases, or other inflammatory diseases comprising administering any antibody (or antibody composition) that specifically binds HMG2 or antigenic fragment thereof (e.g., HMG B box) irregardless of the binding affinity of the antibody.
In another embodiment, the present invention includes methods of treating or preventing arthritis, e.g., rheumatoid arthritis, osteoclast-mediated diseases, or other inflammatory diseases comprising administering a combination of antibodies (or antibody composition) that specifically bind HMG1 and/or HMG2 or antigenic fragment thereof (e.g., HMG B box) irregardless of the binding affinity of the antibody.
In still another embodiment of the present invention, the compositions described herein ameliorate the severity of spinal cord injury (SCI) (by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in a human or a rodent SCI model.
In other specific embodiments, the invention is directed to methods of administering and using compositions and antibodies or the invention to treat and prevent a wide range of inflammatory conditions including both chronic and acute conditions, such as appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cysts, bums, dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac disease, congestive heart failure, restenosis, COPD adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Retier's syndrome, and Hodgkins disease.
The present invention is also directed to a method of inhibiting release of a proinflammatory cytokine from a mammalian cell. The method comprises treating the cell with an antibody or antibody composition of the present invention in an amount sufficient to inhibit release of the proinflammatory cytokine from the cell. In these embodiments, the cell is any cell capable of releasing a proinflammatory cytokine including but not limited to, peripheral blood monocytes and macrophages. In addition, the proinflammatory cytokine may be selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. In a specific embodiment, the cell is a macrophage and the proinflammatory cytokine is selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. In one embodiment, the methods are used to treat a cell in a patient suffering from, or at risk for, a condition characterized by activation of the inflammatory cytokine cascade. Specific conditions are enumerated herein.
In related embodiments, the present invention is directed to a method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade. The method comprises administering to the patient an antibody or an antibody composition of the present invention. Specific conditions have already been enumerated.
Table 1—Antibody Characteristics and Deposit Info
Table 2—Families of Conservative Amino Acid Substitutions
Table 3—Legend for Sequence Listing
Table 4—Legend for Passive CIA mouse model treatment protocol
Table 5—Legend for AIA Rat model treatment protocol
Table 6—Legend for Peritonitis model treatment protocol
The present invention is based in part of the discovery of antibodies that specifically bind HMG1 (also referred to herein as “HMGB1”) and antigenic fragments thereof which exhibit certain biochemical, binding, and functional characteristics. The antibodies that specifically bind HMG1 are specifically referred to herein as “high affinity antibodies of the invention,” “high affinity antibodies” and are also encompassed by the more expansive terms “antibodies of the invention,” “anti-HMG1 antibodies” and simply “HMG antibodies,” as well as like terms. The present invention is also based in part by the discovery of antibodies that bind both HMG1 and HMG2.
The biochemical characteristics of the antibodies of the invention include but are not limited to, isoelectric point (pI) and melting temperature (Tm). The binding characteristics of the antibodies of the invention include, but are not limited to, binding specificity, dissociation constant (Kd), epitope, ability to distinguish between various forms and/or preparations of HMG1 (e.g., recombinant, native, acetylated) and ability to bind soluble and/or immobilized antigen. The functional characteristics of the antibodies of the present invention include, but are not limited to, inhibition of HMG1-induced cytokine release, inhibition of HMG1 binding to one or more receptor, inhibition of HMG1 binding to the cell surface and protection in one or more model of inflammatory disease (e.g., sepsis, arthritis, acute lung injury, peritonitis).
The antibodies of the invention and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute inflammatory diseases and disorders including, but not limited to, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection.
In addition, the high affinity antibodies of the present invention are useful for diagnostic applications. Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the HMG1 and/or HMG2 polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the HMG1 and/or HMG2 polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
6.1 Antibodies of the Invention
High affinity antibodies or fragments thereof that “specifically bind to HMG1 and antigenic fragments thereof” as used herein refers to, for example, high affinity antibodies or fragments thereof that specifically bind to an HMG1 polypeptide or a fragment of an HMG1 polypeptide (e.g., HMG1 A box and HMG1 B Box) or an epitope of an HMG1 polypeptide (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding) and do not specifically bind to other polypeptides. In one embodiment, high affinity antibodies or fragments that specifically bind to an HMG1 polypeptide or fragment thereof do not non-specifically cross-react with other antigens (e.g., binding cannot be competed away with a non-HMG1 protein, e.g., BSA). The present invention also encompasses high affinity antibodies or fragments thereof that specifically bind to an HMG2 polypeptide or fragment of an HMG2 polypeptide (e.g. HMG2 A box and HMG2 B Box) or an antigenic fragment of an HMG2 polypeptide.
It will be recognized by one skilled in the art that HMG1 and HMG2 while being distinct proteins do have regions of homology (see,
In one embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an A box of HMG1 and/or HMG2 (e.g., SEQ ID NOS. 3 and 22, respectively).
In another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a B box of HMG1 and/or HMG2 (e.g., SEQ ID NOS. 4 and 23, respectively).
Also encompassed by the present invention are antibodies which specifically bind to an epitope comprising, or alternatively consisting of (or consisting essentially of) amino acid residues derived from both the A box and B box of HMG1 and/or HMG2. An epitope derived from amino acid residues derived from both the A box B box may be a linear polypeptide derived from the junction of the A and B boxes or may result from the three dimensional confirmation of a polypeptide comprising amino acid residues from both the A and B boxes.
The present invention also specifically encompasses antibodies with multiple specificities (e.g., an antibody with specificity for two or more discrete antigens (reviewed in Cao et al., 2003, Adv Drug Deliv Rev 55:171; Hudson et al., 2003, Nat Med 1:129)). For example, bispecific antibodies contain two different binding specificities fused together. In the simplest case a bispecific antibody would bind to two adjacent epitopes on a single target antigen, such an antibody would not cross-react with other antigens (as described supra). Alternatively, bispecific antibodies can bind to two different antigens, such an antibody specifically binds to two different molecules (e.g., HMG1 and HMG2) but not to other unrelated molecules (e.g., BSA). In addition, an antibody that specifically binds HMG1 and/or HMG2 may cross-react with related HMG proteins.
The term HMG1 and/or HMG2 “fragments” described herein include an HMG1 and/or HMG2 peptide or polypeptide comprising, or alternatively consisting of (or consisting essentially of) an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of HMG1 and/or HMG2 polypeptide (e.g., human HMG1 and/or HMG2).
The term HMG1 and/or HMG2 “fragments” described herein also specifically include polypeptides comprising, or alternatively consisting of (or consisting essentially of) an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, or at least contiguous 80 amino acid residues of an HMG A (e.g., a human HMG1 and/or HMG2 A box) box or an HMG B (e.g., a human HMG1 and/or HMG2 B box) box.
“High affinity antibodies” (also referred to herein as “high affinity antibodies of the invention,” “high affinity antibodies,” and are also encompassed by the more expansive terms “antibodies of the invention” and simply “HMG antibodies”) of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by the instant invention. Examples of BsAbs include without limitation those with one arm directed against an HMG1 and/or HMG2 epitope and the other arm directed against any other antigen. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., 1983, Nature, 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., 1991, EMBO J., 10:3655-3659. A more directed approach is the generation of a Di-diabody a tetravalent bispecific antibody. Methods for producing a Di-diabody are known in the art (see e.g., Lu et al., 2003, J Immunol Methods 279:219-32; Marvin et al., 2005, Acta Pharmacolical Sinica 26:649).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when, the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm (e.g., an HMG1 and/or HMG2 epitope such as the A-box, B-box), and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., 1986, Methods in Enzymology, 121:210. According to another approach described in WO96/27011, a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089) Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Antibodies with more than two valencies incorporating at least one hinge modification of the invention are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
Other antibodies specifically contemplated are “oligoclonal” antibodies. As used herein, the term “oligoclonal” antibodies” refers to a predetermined mixture of distinct monoclonal antibodies. Methods for generating oligoclonal antibodies are known in the art. See, e.g., “Examples Section”, example 1, PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In certain embodiments, oligoclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., HMG1). Those skilled in the art will know or can determine what type of antibody or mixture of antibodies is applicable for an intended purpose and desired need. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to an HMG1 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-HMG1 antibody). It is also specifically contemplated that the antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to an HMG2 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-HMG2 antibody). The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
The antibodies of the invention also encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. As outlined herein, the terms “antibody” and “antibodies” include the antibodies which specifically bind HMG1 and/or HMG2 described herein, full length antibodies and Fc variants thereof comprising Fc regions, or fragments thereof, comprising at least one novel amino acid residue described herein fused to an immunologically active fragment of an immunoglobulin or to other proteins as described herein. Such variant Fc fusions include but are not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) -Fc fusions, scFv-scFv-Fc fusions. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
Antibodies of the present invention also encompass antibodies that have half-lives (e.g., serum half-lives) in a mammal, (e.g., a human), of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the antibodies of the present invention in a mammal, (e.g., a human), results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 and U.S. Patent Publication No. 2003/0190311 and discussed in more detail below).
In one embodiment, the antibodies of the invention may comprise modifications/substations and/or novel amino acids within their Fc domains such as, for example, those disclosed in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, Mol Inmunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Inmunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. patent application Ser. Nos. 10/370,749 and PCT Publications WO 94/2935; WO 99/58572; WO 00/42072; WO 02/060919, WO 04/029207. Other modifications/substitutions of the Fc domain will be readily apparent to one skilled in the art.
Antibodies of the invention comprising modifications/substations and/or novel amino acid residues in their Fc regions can be generated by numerous methods well known to one skilled in the art. Non-limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the variable regions of an antibody of the invention may be subcloned into a vector encoding an Fc region comprising one or modifications/substations and/or novel amino acid residues.
Antibodies of the invention may also be modified to alter glycosylation, again to alter one or more functional properties of the antibody.
In one embodiment, the glycosylation of the antibodies of the invention is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861.
Additionally or alternatively, an antibody of the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342.
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding an HMG1 and/or HMG2 polypeptide or fragment thereof and/or generating a desired response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. See below, Section 6.5 entitled “Antibody Derivitives and Conjugates.”
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides (and polypeptide fragments) with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a human HMG1 polypeptide (e.g., a human HMG1 A box or B box) of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human HMG1 proteins and the corresponding epitopes thereof. Also encompassed by the present invention are antibodies that bind polypeptides (and polypeptide fragments) with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a human HMG2 polypeptide (e.g., a human HMG2 A box or B box). It is further contemplated that antibodies that bind to a human HMG2 polypeptide or fragment thereof may cross-react with murine, rat and/or rabbit homologs of human HMG1 proteins and the corresponding epitopes thereof.
Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to an HMG1 and/or HMG2 polypeptide of the present invention are also included in the present invention.
In one embodiment, antibodies or fragments thereof that specifically bind to an HMG1 polypeptide or fragment thereof prevent/antagonize/inhibit one or more of the following: HMGB1 binding to RAGE, HMGB1 binding to one or more Toll-like receptor (e.g., TLR2 and TLR4), HMGB1 binding to a cell surface (e.g., a THP-1 cell), HMG1-mediated release of proinflammatory cytokines, HMG1-mediated inflammation, HMG1-mediated sepsis, HMG1-mediated inflammation (e.g., of joints), and HMG1-mediated arthritis. These activities may be assayed by one or many known methods in the art. See, e.g., US20040005316, U.S. Pat. Nos. 6,468,533 and 6,448,223, and Section 7 entitled “Examples” infra.
The term “inhibitory concentration 50%” (abbreviated as “IC50”) represents the concentration of an inhibitor (e.g., an antibody of the invention) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets (e.g., HMG1). It will be understood by one in the art that a lower IC50 value corresponds to a more potent inhibitor. In one embodiment, the antibodies of the invention inhibit HMG1-mediated release of proinflammatory cytokines with an IC50 of less than 5000 ng/ml, or of less than 4000 ng/ml, or of less than 3000 ng/ml, or of less than 2000 ng/ml, or of less than 1000 ng/ml, or of less than 500 ng/ml, or of less than 250 ng/ml, or of less than 100 ng/ml, or of less than 50 ng/ml, or of less than 10 ng/ml, or of less than 5 ng/ml. In another embodiment, the antibodies of the invention inhibit HMG1-mediated release of proinflammatory cytokines with an IC50 of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM.
In one embodiment, the antibodies of the invention prevent/antagonize/inhibit the binding of HMG1 to RAGE, but does not substantially affect the binding of HMG1 to one or more of the Toll-like receptors (e.g., TLR2 and TLR4). In another embodiment, the antibodies of the invention prevent/antagonize/inhibit the binding of HMG1 to one or more of the Toll-like receptors (e.g., TLR2 and TLR4), but does not substantially affect the binding of HMG1 to RAGE. In still another embodiment, the antibodies of the invention prevent/antagonize/inhibit the binding of HMG1 to RAGE and inhibit the binding of HMG1 to one or more of the Toll-like receptors (e.g., TLR2 and TLR4). These activities may be assayed by one or many known methods in the art. See, e.g., US20040005316, U.S. Pat. Nos. 6,468,533 and 6,448,223, and Section 7 entitled “Examples” infra.
In a specific embodiment, the antibodies of the invention inhibit the binding of HMG1 to RAGE by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%. In another specific embodiment, the antibodies of the invention inhibit the binding of HMG1 to one or more of the Toll-like receptors (e.g., TLR2 and TLR4) by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by 100%. In another specific embodiment, the antibodies of the invention inhibit the binding of HMG1 to RAGE by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by 100%. In another specific embodiment, the antibodies of the invention inhibit the binding of HMG1 to one or more of the Toll-like receptors (e.g., TLR2 and TLR4) by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by 100%.
As demonstrated herein, antibodies may discriminate between the same polypeptide isolated from different sources. Without wishing to be bound by any particular theory, a polypeptide of similar or identical amino acid sequence isolated from different sources may be distinguished by a number of differences including but not limited to, posttranslational modifications (e.g., phosphorylation, acetylation, methylation, glycosylation, etc.), alterations in overall structure (e.g., changes in disufide bonding and/or folding) and differences in any other molecules that the polypeptide may be associated with (e.g., salts, additional subunits such as polynucleotides and/or other polypeptides). In one embodiment, the antibodies of the invention specifically bind HMG1 recombinantly produced in E. coli with the same or higher affinity than native HMG1 (e.g., isolated from a mammalian cell or tissue). In another embodiment, the antibodies of the invention bind native HMG1 (e.g., isolated from a mammalian cell or tissue) with the same or higher affinity than recombinant HMG1 produced in E. coli. In still another embodiment, the antibodies of the invention will bind one or more forms of native HMG1 including, but not limited to, nuclear HMG1 (e.g., isolated from cells by freeze thaw), released HMG1 (e.g., isolated from the supernatant of necrotic cells) and activated HMG1 (e.g., isolated from stimulated cells, such as LPS stimulated cells). In yet another embodiment, the antibodies of the invention will not bind one or more forms of native HMG1 including, but not limited to, nuclear HMG1 (e.g., isolated from cells by freeze thaw), released HMG1 (e.g., isolated from the supernatant of necrotic cells) and activated HMG1 (e.g., isolated from stimulated cells, such as LPS stimulated cells). Specific methods for obtaining native and recombinant HMG1 are disclosed herein (see Example 2, infra).
In one embodiment, the antibodies of the invention specifically bind to soluble HMG1 and/or HMG2. In another embodiment, the antibodies of the invention specifically bind to immobilized HMG1 and/or HMG2. In still another embodiment, the antibodies of the invention specifically bind to both soluble and insoluble HMG1 and/or HMG2.
As described above, HMG1 and HMG2 are known polynucleotide (i.e. DNA and RNA) binding proteins. In one embodiment, the antibodies of the invention bind to HMG1 and/or HMG2 wherein said HMG1 and/or HMG2 is bound to a polynucleotide molecule. In another embodiment, the antibodies of the invention prevent/antagonize/inhibit one or more of the following: HMGB1 binding to RAGE, HMGB1 binding to one or more Toll-like receptor (e.g., TLR2 and TLR4), HMGB1 binding to a cell surface (e.g., a THP-1 cell), HMG1 -mediated release of proinflammatory cytokines, HMG1-mediated inflammation, HMG1-mediated sepsis, HMG1-mediated inflammation (e.g., of joints), and HMG1-mediated arthritis, wherein said HMG1 is bound to a polynucleotide. In specific embodiments, the polynucleotide molecule is one that promotes an inflammatory response (e.g., a polynucleotide derived from a microorganism or a necrotic cell).
In a specific embodiment, the antibodies of the invention bind acetylated HMG1 with a higher affinity than non-acetylated HMG1. In another specific embodiment, the antibodies of the invention bind non-acetylated HMG1 with a higher affinity than acetylated HMG1. In still another specific embodiment, the antibodies of the invention prevent bind both acetylated and non-acetylated HMG1 with substantially the same affinity.
In another embodiment, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG1 polypeptide or an antigenic fragment thereof of a human or other animal, e.g., mammals and invertebrates. In a specific embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a human HMG1 polypeptide (SEQ ID NO: 1 or 2). HMG1 polypeptides of human and other animals are well known in the art (see, e.g., US20040005316, U.S. Pat. Nos. 6,468,533 and 6,448,223).
In a embodiment, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG1 polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG1 polypeptide of SEQ ID NO:1 or 2.
In another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG1 A box polypeptide of SEQ ID NO:3.
In yet another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG1 B box polypeptide of SEQ ID NO:4 and/or SEQ ID NO: 28 and/or SEQ ID NO:29.
In another embodiment, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG2 polypeptide or an antigenic fragment thereof of a human or other animal, e.g., mammals and invertebrates. In a specific embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a human HMG2 polypeptide (SEQ ID NO:21). HMG2 polypeptides of human and other animals are well known in the art. See, e.g., Jantzen et al., 1990, Nature 344:830-6; Kolodrubetz 1990, Nucleic Acids Res. 18:5565; Laudet et al., 1993, Nucleic Acids Res. 21:2493-501 and Thomas et al., 2001, Trends Biochem Sci. 26:167-74.
In one embodiment, the high affinity antibodies of the invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) an HMG2 polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG2 polypeptide of SEQ ID NO:21.
In another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG2 A box polypeptide of SEQ ID NO:22.
In yet another embodiment, the high affinity antibodies of the present invention specifically bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human HMG2 B box polypeptide of SEQ ID NO:23.
The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined, for example, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A specific, non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al., Nucleic Acids Res., 29:2994-3005 (2001). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, as available on Apr. 10, 2002. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
Another, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl. Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.accelrys.com, as available on Aug. 31, 2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.cgc.com), using a gap weight of 50 and a length weight of 3.
Another embodiment of present invention are antibodies that specifically bind HMG1 and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10−5 M, or of less than 10−6 M, or of less than 10−7 M, or of less than 10−8 M or of less than 10−9 M, or of less than 10−10 M, or of less than 10−11 M, or of less than 10−12 M, or of less than 10−13 M, or of less than 5×10−13M, or of less than 10−14M, less than 5×10−14M, or of less than 10−15M, or of less than 5×10−15M. In still another embodiment, an antibody of the invention that specifically binds HMG1 and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between about 10−7M and about 10−8M, between about 10−8M and about 10−9M, between about 10−9M and about 10−10M, between about 10−10M and about 10−11M, between about 10−11M and about 10−12M, between about 10−12M and about 10−13M, between about 10−13M and about 10−14M. In still another embodiment, an antibody of the invention that specifically binds HMG1 and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between 10−7M and 10−8M, between 10−8M and 10−9M, between 10−9M and 10−10M, between 10−10M and 10−11M, between 10−11M and 10−12M, between 10−12M and 10−13M, between 10−13M and 10−14M.
Another embodiment of present invention are antibodies that specifically bind HMG2 and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10−5 M, or of less than 10−6 M, or of less than 10−7 M, or of less than 10−8 M, or of less than 10−9 M, or of less than 10−10 M, or of less than 10−11M, or of less than 10−12 M, or of less than 10−13 M, or of less than 5×10−13M, or of less than 10−14M, less than 5×10−14M, or of less than 10−15M, or of less than 5×10−15M. In still another embodiment, an antibody of the invention that specifically binds HMG2 and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between about 10−7M and about 10−8M, between about 10−8M and about 10−9M, between about 10−9M and about 10−10M, between about 10−10M and about 10−11M, between about 10−11M and about 10−12M, between about 10−12M and about 10−13M, between about 10−13M and about 10−14M. In still another embodiment, an antibody of the invention that specifically binds HMG2 and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between 10−7M and 10−8M, between 10−8M and 10−9M, between 10−9M and 10−10M, between 10−10M and 10−11M, between 10−11M and 10−12M, between 10−12M and 10−13M, between 10−13M and 10−14M.
It is well known in the art that the equilibrium dissociation constant (Kd) is defined as koff/kon. It is generally understood that a binding molecule (e.g., and antibody) with a low Kd (i.e., high affinity) is preferable to a binding molecule (e.g., and antibody) with a high Kd (i.e., low affinity). However, in some instances the value of the kon or koff may be more relevant than the value of the Kd. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. In certain embodiments, the antibodies of the invention have a lower Kd for one antigen than for others.
In another embodiment, the antibody binds to HMG1 and antigenic fragments thereof with a koff of less than 1×10−3 s−1, or of less than 3×10−3 s−1. In other embodiments, the antibody binds to HMG1 and antigenic fragments thereof with a koff of less than 10−3 s−1, less than 5×10−3 s−1, less than 10−4 s−1, less than 5×10−4 s−1, less than 10−5 s−1, less than 5×10−5 s−1, less than 10−6 s−1, less than 5×10−6 s−1, less than 10−7 s−1less than 5×10−7 s−1, less than 10−8 s−1, less than 5×10−8 s−1, less than 10−9 s−1, less than 5×10−9 s−1, or less than 10−10 s−1.
In another embodiment, the antibody binds to HMG2 and antigenic fragments thereof with a koff of less than 1×10−3 s−1, or of less than 3×10−3 s−1. In other embodiments, the antibody binds to HMG2 and antigenic fragments thereof with a Koff of less than 10−3 s−1, less than 5×10−3 s−1, less than 10−4 s−1, less than 5×10−4 s−1, less than 10−5 s−1, less than 5×10−5 s−1, less than 10−6 s−1, less than 5×10−6 s−1, less than 10−7 s−1, less than 5×10−7 s−1, less than 10−8 s−1, less than 5×10−8 s−1, less than 10−9 s−1, less than 5×10−9 s−1, or less than 10−10 s−1.
In another embodiment, the antibody of the invention binds to HMG1 and/or antigenic fragments thereof with an association rate constant or kon rate of at least 105 M−1s−1, at least 5×105 M−1s−1, at least 106 M−1s−1, at least 5×106 M−1s−1, at least 107 M−1s−1, at least 5×107M−1s−1, or at least 108 M−1s−1, or at least 109 M−1s−1.
In another embodiment, the antibody of the invention binds to HMG2 and/or antigenic fragments thereof with an association rate constant or kon rate of at least 105 M−1s−1, at least 5×105M−1s−1, at least 106 M−1s−1, at least 5×106 M−1 s−1, at least 107 M−1s−1, at least 5×107 M−1s−1, or at least 108 M−1s−1, or at least 109 M−1s−1.
Antibodies like all polypeptides have an Isoelectric Point (pI), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein. As used herein the pI value is defined as the pI of the predominant charge form. The pI of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et al., 1993, Electrophoresis 14:1023). In addition, the thermal melting temperatures (Tm) of the Fab domain of an antibody, can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf-life. A lower Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/more stability. Thus, in certain embodiments antibodies having higher Tm are preferable. Tm of a protein domain (e.g., a Fab domain) can be measured using any standard method known in the art, for example, by differential scanning calorimetry (see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154).
Accordingly, an additional nonexclusive embodiment of the present invention includes high affinity antibodies of the invention that have certain preferred biochemical characteristics such as a particular isoelectric point (pI) or melting temperature (Tm).
More specifically, in one embodiment, the high affinity antibodies of the present invention have a pI ranging from 5.5 to 9.5. In still another specific embodiment, the high affinity antibodies of the present invention have a pI that ranges from about 5.5 to about 6.0, or about 6.0 to about 6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5 to about 8.0, or about 8.0 to about 8.5, or about 8.5 to about 9.0, or about 9.0 to about 9.5. In other specific embodiments, the high affinity antibodies of the present invention have a pI that ranges from 5.5-6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5. Even more specifically, the high affinity antibodies of the present invention have a pI of at least 5.5, or at least 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or at least 6.9, or at least 7.1, or at least 7.3, or at least 7.5, or at least 7.7, or at least 7.9, or at least 8.1, or at least 8.3, or at least 8.5, or at least 8.7, or at least 8.9, or at least 9.1, or at least 9.3, or at least 9.5. In other specific embodiments, the high affinity antibodies of the present invention have a pI of at least about 5.5, or at least about 6.0, or at least about 6.3, or at least about 6.5, or at least about 6.7, or at least about 6.9, or at least about 7.1, or at least about 7.3, or at least about 7.5, or at least about 7.7, or at least about 7.9, or at least about 8.1, or at least about 8.3, or at least about 8.5, or at least about 8.7, or at least about 8.9, or at least about 9.1, or at least about 9.3, or at least about 9.5.
It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pI. For example the pI of a polypeptide can be manipulated by making the appropriate amino acid substitutions (e.g., by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine). Without wishing to be bound by any particular theory, amino acid substitutions of an antibody that result in changes of the pI of said antibody may improve solubility and/or the stability of the antibody. One skilled in the art would understand which amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pI. In one embodiment, a substitution is generated in an antibody of the invention to alter the pI. It is specifically contemplated that the substitution(s) of the Fc region that result in altered binding to FcγR (described supra) may also result in a change in the pI. In another embodiment, substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in FcγR binding and any desired change in pI.
In one embodiment, the high affinity antibodies of the present invention have a Tm ranging from 65° C. to 120° C. In specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from about 75° C. to about 120° C., or about 75° C. to about 85° C., or about 85° C. to about 95° C., or about 95° C. to about 105° C., or about 105° C. to about 115° C., or about 115° C. to about 120° C. In other specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from 75° C. to 120° C., or 75° C. to 85° C., or 85° C. to 95° C., or 95° C. to 105° C., or 105° C. to 115° C., or 115° C. to 120° C. In still other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least about 65° C., or at least about 70° C., or at least about 75° C., or at least about 80° C., or at least about 85° C., or at least about 90° C., or at least about 95° C., or at least about 100° C., or at least about 105° C., or at least about 110° C., or at least about 115° C., or at least about 120° C. In yet other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least 65° C., or at least 70° C., or at least 75° C., or at least 80° C., or at least 85° C., or at least 90° C., or at least 95° C., or at least 100° C., or at least 105° C., or at least 110° C., or at least 115° C., or at least 120° C.
In a specific embodiment, the high affinity antibodies of the invention or fragments thereof are human or humanized antibodies.
In one embodiment, the invention also include particular antibodies (and fragments thereof) that specifically bind HMG1 with high affinity. In particular are the anti-HMG1 antibodies referred to here as “S2”, “S4”, “S16” and “G4” which have been deposited with the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) and assigned ATCC Deposit Nos. PTA-6142, PTA-6143, PTA-6259 and PTA-6258, respectively. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Since the strain referred to is being maintained under the terms of the Budapest Treaty, it will be made available to a patent office signatory to the Budapest Treaty.
In another embodiment, the invention includes antibodies that specifically bind HMG1 and/or HMG2 which comprise one or more of the variable regions disclosed herein (see
The present invention also encompasses variants of G2, G4, G9, G12, G16, G20, G34, G35, S2, S6, S10, S12, S14, S16, S17 and E11 (see
It will be understood that the complementarity determining regions (CDRs) residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Specifically, residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of “spacer” residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al. supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
In other embodiments, the invention includes antibodies having least one, at least two, at least three, at least four, at least five, or at least six of the CDRs disclosed herein (see, e.g.,
The present invention encompasses antibodies that specifically bind to HMG1 and/or HMG2, comprising derivatives of the VH domains, VH CDRs, VL domains, or VL CDRs described herein that specifically bind to HMG1 and/or HMG2. Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an antibody of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions. In one embodiment, the VH and/or VL CDRs derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions in the relative to the original VH and/or VL CDRs. In another embodiment, the VH and/or VL CDRs derivatives have conservative amino acid substitutions (e.g. supra) are made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to HMG1 and/or HMG2). Alternatively, mutations can be introduced randomly along all or part of the VH and/or VL CDR coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.
The present invention also encompasses antibodies that specifically bind to HMG1 and/or HMG2 or a fragment thereof, said antibodies comprising an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of G2, G4, G9, G12, G16, G20, G34, G35, S2, S6, S10, S12, S14, S16, S17 and E11 (see
The present invention further encompasses antibodies that specifically bind to HMG1 and/or HMG2 or a fragment thereof, said antibodies or antibody fragments comprising an amino acid sequence of one or more CDRs that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of one or more CDRs of G2, G4, G9, G12, G16, G20, G34, G35, S2, S6, S10, S12, S14, S16, S17 and E11 (see
The present invention also encompasses antibodies that specifically bind to HMG1 and/or HMG2 or fragments thereof, where said antibodies are encoded by a nucleotide sequence that hybridizes to the nucleotide sequence of G2, G4, G9, G12, G16, G20, G34, G35, S2, S6, S10, S12, S14, S16, S17 and E11 (see
Antibodies having at least one, at least two, at least three, at least four, at least five, or all six of the CDRs of the deposited antibodies are specific embodiments of the invention. Isolated polynucleotides that encode these antibodies (and fragments thereof) are also specific embodiments of the invention. The binding and functional characteristics for “S2”, “S4”, “S16” and “G4” as well as several other specific anti-HMG1 antibodies of the invention are listed in Table 1.
S. aureus
aNo value indicates that the assay was not performed or has not yet been determined
bDetermined via BIAcore analysis using recombinant HMG1 (see Examples section, Example 1), note: the Kd for E11 and S12 have not yet been determined by BIAcore
cDetermined via isoelectric focusing gel electrophoresis (see Examples section, Example 1)
dDetermined via differential scanning calorimetry (see Examples section, Example 1)
fRelative inhibition indicated for mMØ and TLR4, IC50 (nM) indicated for Thp-1 binding (see Examples section, Example 4)
gDetermined by cytokine release inhibition analysis using recombinant HMG1 (see Examples section, Example 3) IC50 values for IL-6 are listed as nM the rest are listed as ng/ml
hPercent protection is calculated by subtracting the isotype control (see Examples section, Examples 5-11).
Another embodiment of the present invention includes the introduction of conservative amino acid substitutions in any portion of an anti-HMG1 antibody of interest, described supra (see table 1). It is well known in the art that “conservative amino acid substitution” refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 2.
The term “conservative amino acid substitution” also refers to the use of amino acid analogs or variants. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” (1990, Science 247:1306-1310).
6.2 Methods of Generating and Screening Antibodies of the Invention
High affinity antibodies or fragments that specifically bind to an HMG1 polypeptide can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, an HMG1 polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immnunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
A “monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
The antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Betteret al., Science 240:1041-1043 (1988).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).
For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397. Humanized antibodies are antibody molecules from non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
If the antibody is used therapeutically in in vivo applications, the antibody is preferably modified to make it less immunogenic in the individual. For example, if the individual is human the antibody is preferably “humanized”; where the complementarity determining region(s) of the antibody is transplanted into a human antibody (for example, as described in Jones et al., Nature 321:522-525, 1986; and Tempest et al., Biotechnology 9:266-273, 1991).
Phage display technology can also be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-B box antibodies or from naive libraries (McCafferty et al., Nature 348:552-554, 1990; and Marks, et al., Biotechnology 10:779-783, 1992). The affinity of these antibodies can also be improved by chain shuffling (Clackson et al., Nature 352: 624-628, 1991).
The choice of polypeptide to be used for the generation can be readily determined by one skilled in the art. Polypeptides may be chosen such that the antibody generated will not significantly cross-react or specifically bind to another member of the HMG protein family. Alternatively, polypeptides which share a large degree of homology between two or more members of the HMG protein family may be used for the generation of an antibody that can specifically bind (i.e., cross-react) with multiple members of the HMG protein family (e.g., HMG1 and HMG2).
6.3 Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide sequence encoding a high affinity antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode an antibody that specifically binds to an HMG1 and/or HMG2 polypeptide of the invention (e.g. SEQ ID NO: 1 or 2 or fragments thereof). In a particular embodiment, a polynucleotide of the invention encodes an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 2. In another embodiment, a polynucleotide of the invention encodes an antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO:3. In another preferred embodiment, a polynucleotide of the invention encodes an antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO:4. In another embodiment, a polynucleotide of the invention encodes an antibody which binds a polypeptide having the amino acid sequence of SEQ ID NO:21. In still another embodiment, a polynucleotide of the invention encodes an antibody which binds a polypeptide having the amino acid sequence of SEQ ID NO:22. In yet another embodiment, a polynucleotide of the invention encodes an antibody which binds a polypeptide having the amino acid sequence of SEQ ID NO:23. In still other embodiments, a polynucleotide of the invention encodes an antibody which binds a polypeptide having the amino acid sequence of SEQ ID NO:28 and/or 29.
By “stringent hybridization conditions” is intended overnight incubation at 42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium cirate), 50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1.times.SSC at about 65.degree. C.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains of the antibodies of the invention may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
6.4 Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, 3T3, PerC6 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)). Also see, e.g., U.S. Pat. Nos. 5,827,739, 5,879,936, 5,981,216, and 5,658,759.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, NS0, Per.C6 and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981).
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell Biol. 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
6.5 Antibody Conjugates and Derivatives
The antibodies of the invention include derivatives that are modified (e.g., by the covalent attachment of any type of molecule to the antibody). For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
Further, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half life in vivo. The techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413,622. The present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
In one embodiment, the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, specifically to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. In another embodiment, the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, specifically to a polypeptide of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.
The present invention further includes formulations comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.
Additional fusion proteins, e.g., of antibodies that specifically bind HMG1 and/or HMG2 or fragments thereof (e.g., supra), may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313. Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to a C/CLP may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Moreover, the antibodies of the invention or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include but are not limited to, 125I, 131I, 111In or 99Tc, in addition positron emitting metals using various positron emission tomographies, noradioactive paramagnetic metal ions, and molecules that are radiolabelled or conjugated to specific radioisotopes can be conjugated to the antibodies of the invention.
Further, an antibody of the invention or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213 Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT publications WO 03/075957/
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), CD40 Ligand, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody in Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
The antibodies of the invention can be conjugated to other polypeptides. Methods for fusing or conjugating antibodies to polypeptide moieties are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995, J Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:11337. The fusion of an antibody to a moiety does not necessarily need to be direct, but may occur through linker sequences. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
6.6 Assays for Antibody Binding and Activity
The antibodies of the invention may be assayed for specific (i.e., immunospecific) binding by any method known in the art. The immunoassays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree. C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4.degree. C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity and other binding properties (e.g., off-rate of an antibody-antigen interaction) of an antibody to an antigen may be determined by a variety of in vitro assay methods well known in the art including for example, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel, electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody. Other specific methods are disclosed herein, see Examples 2-4, infra.
The antibodies of the invention may be assayed for biological activity by any method known in the art.
The protocols and formulations of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific therapeutic protocol formulation or combination therapy of the invention is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise contacted with a formulation of the invention, and the effect of such a formulation upon the tissue sample is observed. The tissue sample can be obtained by biopsy from the patient. This test allows the identification of the therapeutically most effective prophylactic or therapeutic agent(s) for each individual patient. In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in an autoimmune disorder, an inflammatory disorder, a disorder associated with aberrant expression and/or activity of HMG1 and/or HMG2, to determine if a formulation of the invention has a desired effect upon such cell types. For example, a lower level of HMG1 and/or HMG2 and/or proinflammatory cytokines produced by the contacted cells indicates that the composition of the invention may be effective to treat the condition in the patient. Alternatively, instead of culturing cells from a patient, a formulation of the invention may be screened using cells which can be stimulated by HMG1 and/or HMG2 such as for example peripheral blood mononuclear cells (PBMCs), THP-1 cells or Macrophages (MØs). Many assays standard in the art can be used to assess cytokine production including ELISA assays, realtime PCR and other methods well known in the art. Specific methods are also disclosed herein (see Examples 2 and 6, infra).
Prophylactic or therapeutic agents can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc.
The principle animal models for known in the art and widely used are known and described in the art as described above. In addition, specific animal models for sepsis (see Example 5), peritonitis (see Example 10) and arthritis (see Examples 7-9) are disclosed herein.
Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the combinatorial therapies disclosed herein for treatment or prevention of inflammatory disease.
6.7 Antibodies, Antibody Compositions of the Invention and Therapeutic and/or Prophylactic Administration Thereof
The present invention encompasses anti-HMG1 antibodies as disclosed herein and is also directed to antibody compositions referred to herein as “antibody compositions of the invention,” “compositions of the invention” or more simply as “compositions”. In certain embodiments, the compositions of the invention comprise an antibody of the invention in a pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically-acceptable carrier” means a chemical composition with which an antibody of the invention may be combined and which, following the combination, can be used to administer the antibody of the invention to a subject. These antibody compositions are also referred to herein as “pharmaceutical compositions”. In other embodiments, the compositions of the present invention.
The present further includes methods for treating a condition characterized by activation of the inflammatory cytokine cascade, including both acute and chronic inflammatory conditions, comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition of the invention. Chronic inflammatory conditions are charactized by an inflammatory response of prolonged duration—weeks, months, or even indefinitely which results in tissue damage that is often permanent. Chronic inflammatory conditions include but are not limited to, arthritis (e.g. rheumatoid arthritis), inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), cholecystitis. Acute inflammatory conditions are usually charactized by a sudden onest of symptoms including, increased vascular permeability, oedema, systemic fever often resulting in tissue necrosis and may result in death. Acute inflammatory conditions include but are not limited to, sepsis (e.g., due to microbial infection), hypersensitivity reactions, tissue necrosis and appendicitis. The condition can be one where the inflammatory cytokine cascade causes a systemic reaction, such as with endotoxic shock. Alternatively, the condition can be mediated by a localized inflammatory cytokine cascade, as in rheumatoid arthritis.
In one embodiment, compositions of the invention comprise high affinity antibodies that specifically bind to an A box of HMG1 (e.g., an epitope within SEQ ID NO: 3). In another embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to a B box of HMG1 (e.g., an epitope within SEQ ID NOS: 4, 28, 29). In still another embodiment, compositions of the invention comprise high affinity antibodies that specifically bind to an A box of HMG2 (e.g., an epitope within SEQ ID NOS: 22). In another embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to a B box of HMG2 (e.g., an epitope within SEQ ID NOS: 23).
As described above, HMG1 signaling is mediated, at least in part, via the RAGE and via members of the TLR family of proteins. Both the A box and B box likely play a role in receptor binding and signaling. Without wishing to be bound by any particular theory it is therefore contemplated that a combination of antibodies (or other antagonists) which specifically bind the A box and antibodies (or other antagonists) which specifically bind the B box would effectively block HMG1 binding to RAGE and/or TLR proteins. Accordingly, compositions of the invention may comprise a combination of high affinity antibodies of the invention (or other HMGB1 antibodies or antagonists), for example, but not by way of limitation, a combination of antibodies that specifically bind to an HMG1 A box and antibodies that specifically bind a HMG1 B box, or a combination of antibodies that specifically bind to an HMG2 A box and antibodies that specifically bind a HMG2 B box. In a specific embodiment, compositions comprise high affinity antibodies of the invention that specifically bind to an epitope derived from both the A box and B box of HMG1 (e.g., an epitope which spans the junction between the A and B box).
Compositions of the invention can comprise the high affinity antibodies of the present invention alone or in combination with other active therapeutic molecules and/or adjuvants such as steroids, other anti-inflammatory molecules, or other antibody therapeutics. More specifically, the compositions of the invention can comprise an antagonist of an early sepsis mediator. The antagonist of an early sepsis mediator is in one embodiment, an antagonist of a cytokine selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. In a specific embodiment, the antagonist of an early sepsis mediator is an antibody to TNF or MIF, or an IL-1 receptor antagonist.
In one embodiment the pharmaceutical compositions of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, even trace amounts of harmful and dangerous endotoxin must be removed. In certain specific embodiments, the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
When used for in vivo administration, the compositions described herein should be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes or by other means well known in the art. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington's Pharmaceutical Sciences (180′ ed, Mack Publishing Company, Easton, Pa., 1990). Compositions comprising antibodies, such as those disclosed herein, ordinarily will be stored in lyophilized form or in solution. It is contemplated that sterile compositions comprising antibodies of the invention are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
In one embodiment of the present invention, the compositions described herein can inhibit a condition mediated or characterized by activation of an inflammatory cytokine cascade including both acute and chronic inflammatory conditions. In a specific embodiment, the compositions described herein are useful for the treatment of an acute inflammatory condition (e.g., sepsis). In another specific embodiment, the compositions described herein are useful for the treatment of a chronic inflammatory condition (e.g., rheumatoid arthritis). In yet another embodiment, the compositions described herein are useful for the treatment of both acute and chronic inflammatory conditions.
It is contemplated that the compositions of the invention will be protective when administered to a subject having an inflammatory condition mediated or characterized by activation of an inflammatory cytokine cascade. The protection conferred by a composition of the invention may be measured by methods well known in the art. Methods used to determine how protective a composition of the invention is will vary depending on the condition being treated and/or prevented and the measurements be examined. For example, in a rodent model of arthritis a comparison of the paw inflammation scores of animals treated with a composition of the invention and animals treated with an appropriate control composition may be used to determine how protective a composition of the invention is. In a CLP model of sepsis a comparison of the survival of animals treated with a composition of the invention and animals treated with an appropriate control composition may be used to determine protection conferred by antibody treatment. Generally, treatment with a composition of the invention will be compared to certain control treatments. For example, a control treatment may comprise a control antibody or may comprise only excipient. In some cases the control may be the standard of care (or an appropriate surrogate molecule) for treatment of a disease such as for example methotrexate or anti-TNFs (e.g., Enbrel, Humira) for treatment of arthritis. In some cases the control may be a negative control (e.g., PBS). When the control represents the standard of care the composition of the invention may be administered either alone or in combination with the standard of care treatment and the level of protection for each group compared. The choice of control will depend on factors including condition being treated and/or prevented and the measurements be examined and can be readily determined by one of skill in the art. Specific examples of methods to determine the protection conferred by a composition of the invention are provided herein (see Examples 7-11, infra)
In another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition, in an animal CLP sepsis model. In a specific embodiment, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition, in an animal CLP sepsis model selected from the group comprising, a mouse CLP model and a piglet CLP model. In another specific embodiment, the animal CLP model is the mouse CLP model.
In still another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition, in a mouse collagen-induced arthritis model. In a specific embodiment, the mouse collagen-induced arthritis model is the passive collagen-induced arthritis model. In another specific embodiment, the mouse collagen-induced arthritis model is the active collagen-induced arthritis model.
In yet another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than Renbrel® (with or without methotrexate) in a mouse collagen-induced arthritis model. In a specific embodiment, the mouse collagen-induced arthritis model is the passive collagen-induced arthritis model. In another specific embodiment, the mouse collagen-induced arthritis model is the active collagen-induced arthritis model.
In still another embodiment of the present invention, the compositions described herein reduce bone loss and/or cartilage damage (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in a mouse collagen-induced arthritis model. In a specific embodiment, the mouse collagen-induced arthritis model is the passive collagen-induced arthritis model. In another specific embodiment, the mouse collagen-induced arthritis model is the active collagen-induced arthritis model.
In other embodiments, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition in a rat adjuvant-induced arthritis model.
In still other embodiments of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than Renbrel® (with or without methotrexate) in a rat adjuvant-induced arthritis model.
In yet other embodiments of the present invention, the compositions described herein reduce bone loss and/or cartilage damage (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in a rat adjuvant-induced arthritis model.
In another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than Enbrel® (with or without methotrexate) in humans.
In yet another embodiment of the present invention, the compositions described herein are more protective (by at least 10% or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) than a control composition in a mouse peritonitis model.
In still another embodiment of the present invention, the compositions described herein ameliorate the severity of spinal cord injury (SCI) (by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in a human or in a rodent SCI model.
In still another embodiment of the present invention, the compositions described herein ameliorate the severity of acute lung injury (ALI) (by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% ) more than a control composition in a human or in a rodent ALI model.
The present invention is also directed to a method of inhibiting release of a proinflammatory cytokine from a mammalian cell. The method comprises treating the cell with an antibody or antibody composition of the present invention in an amount sufficient to inhibit release of the proinflammatory cytokine from the cell. In these embodiments, the cell is preferably a macrophage. In certain embodiments, the proinflammatory cytokine is selected from the group consisting of TNF, IL1α, IL-1β, MIF and IL-6. In other embodiments, the cell is a macrophage and the proinflammatory cytokine is selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. In still other embodiments, the cell is a PBMC and the proinflammatory cytokine is selected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6. In one embodiment the methods treat a cell in a patient suffering from, or at risk for, a condition characterized by activation of the inflammatory cytokine cascade. Specific conditions are enumerated herein.
The present invention is also directed to a method of the inhibiting release of HMG1 and/or HMG2 from a mammalian cell. The method comprises treating the cell with an antibody or antibody composition of the present invention in an amount sufficient to inhibit release of HMG1 and/or HMG2 from the cell. The methods preferably treat a cell in a patient suffering from, or at risk for, a condition characterized by activation of the inflammatory cytokine cascade. Preferred conditions are enumerated herein.
Methods to determine the inhibition of cytokines, HMG1 and/or HMG2 release can be determined by numerous methods well known in the art such as those described above and disclosed herein (see Examples 2-11, infra).
As used herein, a “therapeutically effective amount,” an “amount sufficient” and like terms refers to that amount of the therapeutic agent, e.g., an HMG1 antibody composition of the invention, sufficient to treat or manage a disease or disorder mediated by HMG1 and/or HMG2. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of the disease, e.g., delay or minimize the severity of a disease. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of an inflammatory disorder. Further, a therapeutically effective amount with respect to a pharmaceutical composition of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management a disease, e.g., an inflammatory disease.
The present invention further provides methods of preventing, managing, treating or ameliorating an inflammatory disorder or one or more symptoms thereof, said methods comprising administering to a subject in need thereof an antibody composition of the invention and/or one or more therapies. Any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of an inflammatory disorder or one or more symptoms thereof can be used in combination with an antibody composition of the invention. Examples of such agents include, but are not limited to, immunomodulatory agents, an anti-angiogenic agents, anti-inflammatory agents and TNF-.alpha. antagonists.
Nonlimiting examples of conditions which can be usefully treated using the antibody compositions, i.e., pharmaceutical compositions of the present invention include those conditions enumerated in the background section of this specification and below. Preferably, the condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, COPD, rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, restenosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Type I diabetes, ankylosing spondylitis, Retier's syndrome, or Hodgkins disease. In more preferred embodiments, the condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, disseminated bacteremia, burns, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection or graft-versus-host disease.
In a preferred embodiment, the invention is directed to methods of administering and using compositions and antibodies or the invention to treat and/or prevent a condition selected from the group consisting of appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, rheumatoid arthritis, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, disseminated bacteremia, bums, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection and graft-versus-host disease. In the most preferred embodiments, the condition is endotoxic shock or allograft rejection. Where the condition is allograft rejection, the composition may advantageously also include an immunosuppressant that is used to inhibit allograft rejection, such as cyclosporin.
A specific preferred embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and/or prevent sepsis and arthritis (e.g., RA, psoriatic arthritis, juvenile rheumatoid arthritis). In addition, a specific preferred embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and prevent psoriasis and ankylosing spondylitis.
Another specific preferred embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and prevent restenosis, vascular diseases, and cardiovascular disease.
Yet another specific preferred embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and prevent tissue damage and to promote tissue repair and regeneration.
As discussed supra, any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of an inflammatory disorder or one or more symptoms thereof can be used in combination with an antibody composition of the invention. Specific examples of immunomodulatory agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1.RTM. (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson)) anti-CD20 antibodies (e.g., Rituxan (IDEC & Genentech, U.S. and International Patent Publications US2004/0202658, WO00/67796) and derivatives there of, HuMax-CD20 (GenMab and Medarex, U.S. Patent Publication 2004/0167319)), anti-CD19 antibodies (see, e.g., U.S. and international Patent Publications US20020041847, US20030133930 and WO 05/012493), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies (e.g., MEDI-507 (MedImmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC)); anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-.alpha. antibodies, anti-IL-.beta. antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies)); anti-CD22 antibodies (e.g., non-ligand blocking antibodies such as Epratuzumab (Immunomedics) and ligand blocking antibodies (e.g., U.S. Patent Publictions 2004/0001828 and 2003/0202975)); CTLA4-immunoglobulin; LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432); soluble cytokine receptors (e.g., the extracellular domain of a TNF-.alpha. receptor or a fragment thereof, the extracellular domain of an IL-1.beta. receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof); cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-.alpha., TNF-.beta., interferon (IFN)-.alpha., IFN-.beta., IFN-.gamma., and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-.alpha. antibodies, and anti-IFN-.gamma. antibodies).
Non-limiting examples of anti-angiogenic agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include Vitaxin® (MedImmune) or other anti-alpha v beta3 antibodies (e.g., CNTO95 (Centocor)), endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of platelet factor-4, the anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the small anti-angiogenic peptides of laminin, fibronectin, procollagen and EGF, acid fibroblast growth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF) antagonists, vascular endothelial growth factor (VEGF) antagonists, VEGF receptor (VEGFR) antagonists (e.g., anti-VEGFR antibodies), and Avastin ®.
Non-limiting examples of TNF-.alpha. antagonists which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab).sub.2 fragments, and antigen-binding fragments thereof) such as antibodies that immunospecifically bind to TNF-.alpha., nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules that blocks, reduces, inhibits or neutralizes the function, activity and/or expression of TNF-.alpha.. In various embodiments, a TNF-.alpha. antagonist reduces the function, activity and/or expression of TNF-.alpha. by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a control such as phosphate buffered saline (PBS). Examples of antibodies that immunospecifically bind to TNF-.alpha. include, but are not limited to, infliximab (REMICADE.™.; Centacor), D2E7 (Abbott Laboratories/Knoll Pharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known as HUMICADE.™. and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), and TN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91: 2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA 89:7375-7379). The present invention also encompasses the use of antibodies that immunospecifically bind to TNF-.alpha. disclosed in the following U.S. patents in the compositions and methods of the invention: U.S. Pat. Nos. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380; 5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272; 5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and 6,171,787. Examples of soluble TNF-.alpha. receptors include, but are not limited to, sTNF-R1 (Amgen), etanercept (ENBREL™; Immunex) and its rat homolog RENBREL™, soluble inhibitors of TNF-.alpha. derived from TNFrI, TNFrII (Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335), and TNF-.alpha. Inh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192).
Other TNF-.alpha. antagonists encompassed by the invention include, but are not limited to, IL-10, which is known to block TNF-.alpha. production via interferon .gamma.-activated macrophages (Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-1 (Serono/Yeda), the vaccine CytoTAb (Protherics), antisense molecule 104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax), auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals), quinacrine (mepacrine dichlorohydrate), tenidap (Enablex), Melanin (Large Scale Biological), and anti-p38 MAPK agents by Uriach.
Non-limiting examples of anti-inflammatory agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-agonists, anticholingeric agents, and methyl xanthines. Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX.™.), diclofenac (VOLTAREN.™.), etodolac (LODINE.™.), fenoprofen (NALFON.™.), indomethacin (INDOCIN.™.), ketoralac (TORADOL.™.), oxaprozin (DAYPRO.™.), nabumentone (RELAFEN.™.), sulindac (CLINORIL.™.), tolmentin (TOLECTIN.™.), rofecoxib (VIOXX.™.), naproxen (ALEVE.™., NAPROSYN.™.), ketoprofen (ACTRON.™.) and nabumetone (RELAFEN.™.). Such NSAIDs function by inhibiting a cyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON.™.), cortisone, hydrocortisone, prednisone (DELTASONE.™.), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes, and leukotrienes.
In specific embodiments, patients with osteoarthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful for osteoarthritis prevention, treatment, management or amelioration including but not limited to: analgesics (non-limiting examples are acetaminophen, in a dose up to 4000 mg/d; phenacetin; and tramadol, in a daily dose in the range of 200 to 300 mg); NSAIDs (non-limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin. Low dose NSAIDs are preferred, e.g., ibuprofen at 1200 mg/d, naproxen at 500 mg/d. A gastroprotective agent, e.g., misoprostol, famotidine or omeprazole, is preferred to use concurrently with a NSAID); nonacetylated salicylates including but not limited to salsalate; cyclooxygenase (Cox)-2-specific inhibitors (CSIs), including but not limited to, celecoxib and rofecoxib; intra- or periarticular injection of a depot glucocorticoid preparation; intra-articular injection of hyaluronic acid; capsaicin cream; copious irrigation of the osteroarthritis knee to flush out fibrin, cartilage shards and other debris; and joint replacement surgery. The antibody compositions of the invention can also be used in combination with other nonpharmacologic measures in prevention, treatment, management and amelioration of osteoarthritis including but not limited to: reduction of joint loading (non-limiting examples are correction of poor posture, support for excessive lumbar lordosis, avoid excessive loading of the involved joint, avoid prolonged standing, kneeling and squatting); application of heat to the affected joint; aerobic exercise and other physical therapies.
In specific embodiments, patients with rheumatoid arthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to: NSAIDs (non-limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.); analgesics (non-limiting examples are acetaminophen, phenacetin and tramadol); CSIs including but not limited to, celecoxib and rofecoxib; glucocorticoids (preferably low-dose oral glucocorticoids, e.g., <7.5 mg/d prednisone, or monthly pulses with high-dose glucocorticoids, or intraarticular glucocorticoids); disease-modifying antirheumatic drugs (DMARDS) including but not limited to, methotrexate (preferably given intermittent low dose, e.g., 7.5-30 mg once weekly), gold compounds (e.g., gold salts), D-penicillamine, the antimalarials (e.g., chloroquine), and sulfasalazine; TNF-.alpha. neutralizing agents including but not limited to, etanercept and infliximab; immunosuppressive and cytotoxic agents (examples include but not limited to, azathioprine, leflunomide, cyclosporine, and cyclophosphamide), and surgery (examples include but not limited to, arthroplasties, total joint replacement, reconstructive hand surgery, open or arthroscopic synovectomy, and early tenosynovectomy of the wrist). The antibody compositions of the invention may also be used in combination with other measures in prevention, treatment, management and amelioration of the rheumatoid arthritis including but not limited to: rest, splinting to reduce unwanted motion of inflamed joint, exercise, used of a variety of orthotic and assistive devices, and other physical therapies. The antibody compositions of the invention may also be used in combination with some nontraditional approaches in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to, diets (e.g., substituting omega-3 fatty acids such as eicosapentaenoic acid found in certain fish oils for dietary omega-6 essential fatty acids found in meat), vaccines, hormones and topical preparations.
In specific embodiments, patients with chronic obstructive pulmonary disease (COPD) are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with other agents or therapies useful in prevention, treatment, management and amelioration of COPD including, but not limited to: bronchodilators including but not limited to, short- and long-acting beta..sub.2-adrenergic agonists (examples of short-acting .beta..sub.2 agonist include but not limited to, albuterol, pirbuterol, terbutaline, and metaproterenol; examples of long-acting .beta..sub.2 agonist include but not limited to, oral sustained-release albuterol and inhaled salmeterol), anticholinergics (examples include but not limited to ipratropium bromide), and theophylline and its derivatives (therapeutic range for theophylline is preferably 10-20 .mu.g/mL); glucocorticoids; exogenous .alpha..sub.1AT (e.g., .alpha..sub.1AT derived from pooled human plasma administered intravenously in a weekly dose of 60 mg/kg ); oxygen; lung transplantation; lung volume reduction surgery; endotracheal intubation, ventilation support; yearly influenza vaccine and pneumococcal vaccination with 23-valent polysaccharide; exercise; and smoking cessation.
In specific embodiments, patients with pulmonary fibrosis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for pulmonary fibrosis therapy including but not limited to: oxygen; corticosteroids (a non-limiting example is to administer daily prednisone beginning at 1-1.5 mg/kg/d (up to 100 mg/d) for six weeks and tapering slowly over 3-6 months to a minimum maintenance dose of 0.25 mg/kg/d); cytotoxic drugs (non-limiting examples are cyclophosphamide at 100-120 mg orally once daily, and azathioprine at 3 mg/kg up to 200 mg orally once daily); bronchodilators (non-limiting examples are short- and long-acting beta..sub.2-adrenergic agonists, anticholinergics, and theophylline and its derivatives); and antihistamines (non-limiting examples are diphenhydramine and doxylamine).
In specific embodiment, patients with SCI are administered prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for SCI therapy including but not limited to: glucocorticoid steroids (a non-limiting example is to administer methylprednisolone 30 mg/kg bolus over 15 minutes and an infusion of methylprednisolone at 5.4 mg/kg/h for 23 hours beginning 45 minutes after the bolus), neuroprotectors (e.g., minocyclin), regeneration therapies (e.g., stem cell treatments, hydrogels), weak electrical fields (e.g., extraspinal oscillating field stimulator implantable medical device).
In specific embodiments, patients with asthma are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for asthma therapy including but not limited to: adrenergic stimulants (examples include but not limited to, catecholamines, e.g., epinephrine, isoproterenol, and isoetharine; resorcinols, e.g., metaproterenol, terbutaline, and fenoterol; and saligenins, e.g., salbutamol. Inhalation is the preferred route of administration for adrenergic stimulants); methylxanthines including but not limited to theophylline and its various salts; anticholinergics including but not limited to, atropine sulfate, atropine methylnitrate, and ipratropium bromide; glucocorticoids (examples including but not limited to systemic or oral steroids, and inhaled glucocorticoids); mast cell stabilizing agents (examples include but not limited to, cromolyn sodium and nedocromil sodium); leukotriene modifiers (examples include but not limited to, Zileuton, zafirlukast and montelukast); immunosuppressant agents (examples include but not limited to, methotrexate and gold salts); and mucolytic agents (examples include but not limited to acetylcysteine).
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Neurol. 25:351; Howard et al.,1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The excipient included with the polypeptide in these compositions is chosen based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as endotoxic shock, and oral administration may be preferred to treat a gastrointestinal disorder such as a gastric ulcer. The route of administration and the dosage of the composition to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. Thus, depending on the condition, the antibody composition can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabuccaly and transdermally to the patient.
Accordingly, compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
The compositions of the present invention can easily be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the antibody compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as, for example, benzyl alcohol or methyl parabens, antioxidants such as, for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Rectal administration includes administering the pharmaceutical compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120C, dissolving the antibody composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves and the like.
The antibody compositions described herein can also include an antagonist of an early sepsis mediator. As used herein, an early sepsis mediator is a proinflammatory cytokine that is released from cells soon (i.e., within 30-60 min.) after induction of an inflammatory cytokine cascade (e.g., exposure to LPS). Nonlimiting examples of these cytokines are TNF, IL-1α, IL-1β, IL-6, PAF, and MIF. Also included as early sepsis mediators are receptors for these cytokines (for example, tumor necrosis factor receptor type 1) and enzymes required for production of these cytokines, for example, interleukin-1β converting enzyme). Antagonists of any early sepsis mediator, now known or later discovered, can be useful for these embodiments by further inhibiting an inflammatory cytokine cascade.
Nonlimiting examples of antagonists of early sepsis mediators are antisense compounds that bind to the mRNA of the early sepsis mediator, preventing its expression (see, e.g., Ojwang et al., 1997, Biochemistry 36:6033-6045; Pampfer et al., 1995, Biol. Reprod. 52:1316-1326; U.S. Pat. No. 6,228,642; Yahata et al., 1996, Antisense Nucleic Acid Drug Dev. 6:55-61; and Taylor et al., 1998, Antisense Nucleic Acid Drug Dev. 8:199-205), ribozymes that specifically cleave the mRNA of the early sepsis mediator (see, e.g., Leavitt et al., 2000, Antisense Nucleic Acid Drug Dev. 10: 409-414; Kisich et al., 1999; and Hendrix et al., 1996, Biochem. J. 314: 655-661), and antibodies that bind to the early sepsis mediator and inhibit their action (see, e.g., Kam and Targan, 2000, Expert Opin. Pharmacother. 1: 615-622; Nagahira et al., 1999, J. Immuno. Methods 222, 83-92; Lavine et al., 1998, J. Cereb. Blood Flow Metab. 18: 52-58; and Holmes et al., 2000, Hybridoma 19: 363-367). Any antagonist of an early sepsis mediator, now known or later discovered, is envisioned as within the scope of the invention. The skilled artisan can determine the amount of early sepsis mediator to use in these compositions for inhibiting any particular inflammatory cytokine cascade without undue experimentation with routine dose-response studies.
6.8 Diagnostics and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen, et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003).
One embodiment of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: (a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; (b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); (c) determining background level; and (d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
Also as described herein, antibodies of the invention may be used to treat, diagnose, or prognose an individual having sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, psoriatic arthritis, arthritis, anaphylactic shock, organ ischemia, reperfusion injury, and allograft rejection.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
anucleotide sequences are designated “nt” amino acid sequences are designated “aa”
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
Development and Physical Characterization of Human anti-HMG1 Antibodies
A large panel of human anti-HMG1 antibodies were isolated from a naïve human Fab phage display library by several rounds of panning against human HMG1 (SEQ ID NO: 1 and 2, also see
7.1.1 Materials and Methods
Isolation of Human anti-HMG1 Antibodies: A human Fab phage display library (Dyax, Cambridge, Mass.) was screened by three rounds of panning as follows: Day 1. I) Coat an immunotube with full length HMGB-1 at 20 μg/ml in 0.1 M Carbonate buffer (pH 9.6). Leave it in 4° C. for overnight. Day 2. I) Using 100 fold of phage as library size. Add ⅕ volume of PEG 6000 (20%). Leave on ice for 1 h. Spin at 14000 rpm for 10 min. Resuspend in PBS (pH 7.4) to precipitate phage library. II) Both the antigen-coated immunotube and the phage are blocked with 2% milk/PBS. The immunotube is then rinsed with PBS 2×. and the phage are transferred to the immunotube and mixed by rotating for 30 min and then allow to incubate for an additional 1.5 hrs stationary. III) The immunotube is washed with PBST (PBS+0.1% Tween 20) 10-20 times then PBS 10-20 times and the phage are eluted with 1 ml of 100 mM triethylamine. Eluted phage are neutralized with 0.5 ml of 1 M Tris-HCl (pH 7.5). IV) 1 volume of eluted neutralized phage are mixed with 5 volumes of log phase TG1 and 4 volume of 2YT. Incubate at 37° C. for 30 min (water bath). The infected cells are harvested by centrifugation and resuspended in 2YT and plated on 2YT agar with carbenicillin and 2% glucose.
Expression of Human Anti-HMG1 Oligoclonal and Monoclonal Antibodies: The plasmid was extracted from several pools of bacterium after 3rd round panning. The Fab gene fragments were then excised from the plasmid and inserted into the IgG expression vector under the control of CMV promoter. The plasmid of IgG expression vector with Fab fragment was transient transfected into 293H cells and the oligoclonal antibodies were purified from grow medium by passing it through protein A column. Each pool of oligoclonal antibodies was tested for reactivity to HMG1. Those pools testing positive were further screen to identify individual positive clones.
Isoelectric Focusing Gel Electrophoresis: Isoelectric points were determined using a Pharmacia Biotech Multiphor 2 electrophoresis system with a multi temp 3 refrigerated bath recirculation unit and an EPS 3501 XL power supply. Pre-cast ampholine gels (Amersham Biosciences, pI range 2.5-10) were loaded with 5 μg of protein. Broad range pI marker standards (Amersham, pI range 3-10, 8 μL) were used to determine relative pI for the Mabs. Electrophoresis was performed at 1500 V, 50 mA for 105 minutes. The gel was fixed using a Sigma fixing solution (5×) diluted with purified water to 1×. Staining was performed overnight at room temperature using Simply Blue stain (Invitrogen). Destaining was carried out with a solution that consisted of 25% ethanol, 8% acetic acid and 67% purified water. Isoelectric points were determined using a Bio-Rad Densitometer relative to calibration curves of the standards.
Differential Scanning Calorimetry: Thermal melting temperatures (Tm) were measured with a VP-DSC (MicroCal, LLC) using a scan rate of 1.0° C./min and a temperature range of 25-120° C. A filter period of 8 seconds was used along with a 5 minute pre-scan thermostating. Samples were prepared by dialysis into 25 mM Histidine-HCl, pH 6 using Pierce dialysis cups (3.5 kD). Average Mab concentrations were 50 μg/mL as determined by A280. Melting temperatures were determined following manufacturer procedures using Origin software supplied with the system. Briefly, multiple baselines were run with buffer in both the sample and reference cell to establish thermal equilibrium. After the baseline was subtracted from the sample thermogram, the data were concentration normalized and fitted using the deconvolution function.
7.1.2 Results
Over 35 individual Fab clones were isolated from a single phage display library, 18 of which were converted into full length IgG1 and purified from transient transfections. The subsequent analysis of these clones demonstrate that they have a wide range of characteristics. For example, they exhibit dissociation constants (Kd) between a high of about 330 nM to a low of just 22 nM (summarized in Table 1). The Tm values, which can give an indication of stability, range from a low of just about 70° C. to a high of about 90° C. (
Kinetic Analysis of Human anti-HMG1 Antibodies
The binding kinetics and specificity of several human anti-HMG1 antibodies was examined using a variety of techniques. In addition, several peptides were used in epitope mapping studies. As summarized in Table 1, these analysis show that the human anti-HMG1 antibodies have differing binding kinetics and specificity. In addition, the data indicate that the anti-HMG1 antibodies bind to a variety of epitopes including the HMG1 B-Box and A-Box.
7.2.1 Materials and Methods
Production/Isolation of Recombinant HMG1: Recombinant HMG1 is purified from E. coli as a calmodulin binding protein (CBP) fusion protein (CBP is fused to N-terminal end of HMGB1). E. coli expressing CBP-HMG1 are induced for 2-3 hours and the protein is release by microfluidization in 25 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, pH 8.0. The lysed cells are centrifuged at 125,000×g for 1 hour, the filtered supernatant is applied to a calmodulin column in the presence of CaCl2. The column is washed with 2-2.5 column volumes of lysis buffer and then with a linear gradient to 50 mM Tris, 400 mM NaCl, 2 mM CaCl2, pH 8.0 in 5 column volumes and the protein is eluted with 100 mM Tris, 400 mM NaCl, 5 mM EGTA, pH 8.0. A TritonX114 extraction is used to remove endotoxin. TX-114 is used at a final concentration of 2% and is incubated at 4C for 30 minutes, moved to 37° C. for 30 minutes and centrifuged to separate the phases. The protein is extracted twice.
Preparation of Three forms of Native HMG1: Nuclear HMG1 is prepared from 293H (ATCC number CRL-1573, human kidney, epithelial) cells grown in DMEM with 10% FBS according to protocol from ATCC. Cells were harvested at 80% confluence by the addition of Trypsin/EDTA for less than 1 min at RT. Cell were recovered at once in PBS by gentle flushing of the flask followed by centrifugation at 1100 rpm for 3 min. Cells were washed twice with PBS and transferred to 2ml eppendorf tube at a final concentration about 2 to 5×107 /ml in PBS and then frozen in liquid nitrogen for 2 minutes followed by a 5-10 minute thaw in water bath at RT. The freeze thaw process was repeated two more times. The lysed cells were centrifuged at 13,000 rpm and the supernatant was removed to a new sterile tube and stored at −70 to −80° C. The amount of supernatant used is based on cell concentration of supernatant prior to freeze thaw.
Released HMG1 is prepared from the conditioned media of necrotic 293H cells. 293H cells were grown in DMEM medium with 10% FBS for 10 days without changing the medium. The medium is harvested from the flask and centrifuged at 3000 rpm for 10 minutes, the supernatant was then passed through a 0.2 um filter and placed into a dialyze bag and dialyzed against concentration solution (PIERCE). The concentration solution was changed as needed until the volume of the media was reduced about ten fold. The concentrated medium was then dialyzed against PBS (pH 7.2). The concentration of HMGB1 present in the concentrated sample was determined by a sandwich ELISA (see below) using a purified HMGB1 as a standard.
Activated HMG1 is prepared from THP-1 (ATCC number TIB-202, human monocyte) cells grown in RPMI1640 (Cat# 03-0078DJ) with 10% FBS, 0.05 mM 2-mercaptoethanol according to protocol from ATCC. Cells were treated with LPS at a final concentration of 0.5 ug/ml overnight (14 to 16 hour) when they reached about 4×105 cells/ml. Cells were collected by centrifugation at 1,100 rpm ×3 min and washed three times with PBS to completely remove media containing LPS and transferred to 2 ml eppendorf tube at a final concentration about 2 to 5×107/ml in PBS and then frozen in liquid nitrogen for 2 minutes followed by a 5-10 minute thaw in water bath at RT. The freeze thaw process was repeated two more times. The lysed cells were centrifuged at 13,000 rpm and the supernatant was removed to a new sterile tube and stored at −70 to −80° C. The amount of supernatant used is based on cell concentration of supernatant prior to freeze thaw.
HMG1 Binding Affinity via BIAcore Analysis: All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Piscataway, N.J.). Briefly, each mAb was immobilized to a CM5 sensor chip using a standard amine coupling chemistry. Separately a reference (control) flow cell was also prepared. Two-fold, serial dilutions of HMG1 in instrument buffer were sequentially injected at a slow flow rate over the individual mAb and reference flow-cell surfaces. Following the binding and dissociation of HMG1, the mAbs surfaces were regenerated with a brief pulse of 1M NaCl-50 mM NaOH. At the end of each experiment, the binding curves were evaluated using a steady-state model available through the BIAevaluation software supplied by BIAcore, Inc. (Piscataway, N.J.). The Kd values determined from these studies are listed in Table 1.
Direct ELISA (immobilized HMG1): Individual wells of a 96-well immunoplate (EIA/RIA plate, High binding, Costar) were coated with HMGB-1 at 5 μg/ml in PBS at 4° C. overnight. The plate was then blocked with 4% milk powder dissolved in PBS at 37° C. for 1 hour. The blocking solution was removed and replaced with human anti-HMG1 antibodies at various concentrations (see
Sandwich ELISA (soluble HMG1): Immunoplates (EIA/RIA plate, High binding, Costar) were coated with anti human IgG Fc at 10 μg/ml in PBS (pH 7.2) and incubated at 4° C. overnight. The coating reagent was removed and the plates were rinsed briefly with PBS. The plates were then blocked with 4% milk for 1 hour at 37° C. and rinsed with PBS. The anti-HMG1 antibodies were diluted in 4% milk. For serial dilutions, the antibodies were used at a starting concentration of 20 μg/ml. The diluted anti-HMG1 antibodies were then added into plate and incubated for 1 hr at 37° C. The plate was then washed 10 times with PBST (PBS/0.1% tween 20) and incubate with antigen (HMGB1, 2 μg/ml; or 0.7 μg/ml for native HMGB1) in 4% milk and incubated at 37° C. for 1 h. The plate was then wash 10 times and incubated with mouse anti-HMGB1, 1 μg/ml in 4% milk at 37° C. for 1 h. The plate was washed 10 times and incubated with anti-mouse IgG-HRP at 1:1000 at 37° C. for 1 h. The plate was then washed and developed. HRP activity was detected with Sureblue HRP substrate (KPL). Plates were read at 450 nm using a Kinetic Microplate Reader (Molecular Devices). The data are summarized in Table 1 and representative binding curves are shown in
Antibody HMG1 Binding Competition via BIAcore Analysis: All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Piscataway, N.J.). The HMGB-1 protein was immobilized onto a CM5 sensor using a standard amine coupling chemistry protocol, as described in the BIAcore Handbook (BIAcore, Inc., Piscataway, N.J.). Briefly, the CM5 surface subject to the NHS/EDC activation. After activation a HMGB-1 was injected over the surface at a concentration of 100 nM or 200 nM (in 10 mM NaOAc, pH4) to a surface density of between 1100 and 1200 RU's. Following this, unreacted sites on the sensor chip surface were “capped” with an injection of 1M ethanolamine. For reference purposes, a blank flow cell was also prepared, utilizing this same procedure that was used to immobilize HMGB-1, but without any ligand.
MAbs G2, G4, G9, S6 and Synagis were prepared at 1 uM and 2 uM. All mAb solutions were prepared in HBS-EP buffer (BIAcore, Inc., Piscataway, N.J.). Each cycle started with a 100 μL injection of the first mAb, injected at 1 μM, followed by a second 100 μL injection of a 1:1 mixture of two, 2× concentration mAbs, such that the final concentration of each component mAb in the mixture is equivalent to the first injection. Following each injection cycle, the HMGB-1 surfaces were regenerated with a 1 min pulse of 10 mM HCl.
Once the entire set had been collected, the maximum RU responses for each mAb after each injection cycle was recorded. These were then used to calculate an average response level for each mAb. This average binding response was then used to calculate the percent each mAb bound to the HMGB-1 surface following it's saturation with the first mAb. Taken together, these binding/blocking patterns were used to determine if the mAbs bound to sites which unrelated or related sites on HMGB-1. These data are summarized in Table 1.
Binding to HMG1 vs. HMG2: Ultra high-binding ELISA plates (ThermoLab systems, #3855) were coated with either 5 ug/mL of Calf thymus HMGB1 or HMBG2 diluted in 10 mM Phosphate buffered saline (PBS), pH 7.2 and incubate overnight at 4 degrees. The plates were washed twice with Ca and Mg free PBS and 100 microliters of anti-HMGB1 antibody diluted in 10 mM PBS, pH 7.2+1% bovine serum albumin (BSA) to a final concentration of 10 ug/mL was added to each well and the plates were incubated 1 hour at room temperature. The wells were washed three times with 100 μl of PBS, pH 7.2. HRP labeled goat-anti-human IgG Kappa-chain (KPL, #14-10-10) and goat-anti-human IgG Lambda-chain (KPL, #14-10-11) were diluted in 10 mM PBS+1% BSA 1:1000 and 100 μl were added to each well and incubated for 1 hour at room temperature. The wells were then washed three times with 100 μl of PBS, pH 7.2. OPD substrate (Pierce chemical company, #34006) was prepared following the manufacturers instructions and 100 μl of prepared substrate was added to each well and the plates incubated at room temperature in the dark for 20-30 minutes. The reaction was stopped with the addition of 100 μl of 2M H2SO4, and the plates read on a plate reader at a wavelength of 490 nm. The OD values for a number of antibodies are shown in
HMG1 B Box Peptide Mapping: Individual wells of a 96-well immunoplate (EIA/RIA plate, High binding, Costar) were coated with HMG1 B Box peptide amino acids 91-169 (
7.2.2 Results
ELISA studies demonstrated that most of the antibodies examined bind to immobilized rHMG1 (
BIAcore Analysis of nearly all of the antibodies was used to determine the Kd of each antibody for recombinant HMG1 (rHMG1, see Table 1). Analysis of several antibodies (G4, G9, S2 and S6) by BIAcore competition assays showed that these anti-HMBG1 antibodies appear to bind to either the same site or sites that are highly related perhaps overlapping (see Table 1).
Peptide mapping studies were done on several of the anti-HMG1 antibodies (S2, S6, S10, G2, G4, G9, S12 and S16) to examine binding to HMG1 B Box. As shown in
In summary, the human anti-HMG1 antibodies isolated show a wide variety of binding characteristics with some binding all forms of HMG1 (e.g., S16) and others discriminating between recombinant and native forms (e.g., S10 and S2). They also bind a number of different epitopes include the A- and B-boxes. Several human anti-HMG1 antibodies were selected for use in a number of addition in vitro and in vivo experiments (see below).
Anti-HMGB1 Antibodies Inhibit Cytokine Release from Human PBMC's
The ability of a panel of human anti-HMG1 antibodies to inhibit HMG1 induced cytokine release from human peripheral blood mononuclear cells (PBMCs) was determined. The effect on the following cytokines were examined, IL-12, IL-1β, TNF-α, and IL-6. A number of anti-HMG1 antibodies are capable of inhibiting the release of one or more of these cytokines induced by HMG1. In addition, it was determined that HMG1 can stimulate the release of NO and that antibodies against HMG1 can inhibit this release. The ability of several human anti-HMG1 antibodies to reduce HMG1 induced cytokine gene expression in mouse macrophages was also demonstrated.
7.3.1 Materials and Methods
Inhibition of Cytokine Release: Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers by a density gradient centrifugation. Freshly drawn heparinized whole blood was mixed with two volume of PBS. The diluted blood was gently layered on the surface of Histopaque-1077 (Sigma-Aldrich) and centrifuged at 400×g, at RT for 30 minute. PBMCs were collected from the interface between the plasma and the density gradient solution. After washing in PBS 3×, the purified PBMCs were resuspended in RPMI-1640 medium (GIBCO BRL) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. 1×105 of cells was added in each well of 96-well cell culture plates.
The PBMCs were incubated for two hours at 37° C. with 5% CO2. recombinant HMG1, at 4 μg/ml, or native activated HMG1 from 2.4×105 LPS stimulated THP-1 cells, and different concentrations of human anti-HMGB-1 monoclonal antibodies, a RAGE-Fc fusion protein or an HMG1 A-box-Fc fusion protein were added into each well. The culture media was supplemented with 8 U/ml (1 μg/ml) Polymyxin B sulphate (Sigma-Aldrich) to inhibit potential endotoxin. PBMCs from the same donor without stimulation with HMG1 were used as controls. The cell-free culture media were harvested after 14 hours and stored at −20° C.
The culture media were analyzed for inflammatory cytokines using Beadlyte human multi-cytokine flex kit from Upstate by Luminex 100 (Luminex Corp.). Proinflammatory cytokines TNF-α, IL-6, IL-1β0 and IL-12 (p40) were measured for cells stimulated with recombinant HMG1. In addition, TNF-α, IL-6, IL-1β and IL-8 were measured for cells stimulated with native activated HMG1.
Cytokine release data is presented as mean of triplicates±standard deviations. IC50 for anti-HMGB1 monoclonal antibody is defined as the concentration of antibody required yielding one-half maximal inhibition of the cytokine release from PBMCs stimulated by HMGB1. It was calculated by PRISM program.
Inhibition of NO Release: Macrophages employed in the NO assay included RAW cells (mouse macrophage cell line), as well as bone marrow-derived macrophages (mBMMf) obtained from C57BL/6 mice. The mBMMf were used after maturing for 3 days in culture in the presence of M-CSF (“fresh mBMMf”). Cells were plated at 105 cells/well into 96-well plates and stimulated with HMG1 over night in 100 μl of serum-free a-medium. HMGB-1 (5 μg/ml) and LPS (1 μg/ml) were used as positive controls to stimulate NO production by the macrophages at various concentrations; dose dependent response observed with these stimuli. Antibodies were tested at a molar ration of 4:1 against HMG1. The next day plates are spun at 1500 rpm for 5 min and the supernatant is harvested. To another 96-well plate the following components are mixed, A 50 μl of stimulated supernatant and standards (diluted in a-medium), 25 μl of NADH, 25 μl of Nitrate Reductase, 50 μl of Griess Reagent I and the plate is incubated for 30 min. at 37° C. Then 50 μl of Griess Reagent II is added to each well and the plate is incubated for 10 minutes at room temp. The absorbance of each well is read at 540 nm and the values of Nitrate are calculated against a standard curve.
Tagman Analysis of Mouse Macrophages (mMØ) Stimulated with HMG1: Mouse bone marrow was harvested by rinsing the femurs of normal C57BL/6 mice. The isolated bone marrow cells were then cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 ng/ml M-CSF for 24 h, nonadherent cells were collected and grown for 6 days in complete DMEM supplemented with 10% FBS and 50 ng/ml M-CSF in T-75 flasks (1×107 cells/15 ml/flask). On day 6, adherent cells were harvested and reseeded with 5 ng/ml MCSF in α-MEM containing 10% FBS in 96-well plates (1×105 cells/200 μl/well) overnight.
The culture medium was replaced with a-MEM and incubated for 2 hours before stimulation to starve cells. HMGB1 (recombinant CBP-HMGB1 fusion protein generated from E. coli at 10 μg/ml), mouse-RAGE-Fc or human-RAGE-Fc fusion protein were pre-mixed with various blocking reagents at 100 μg/ml in α-MEM for 20 min at RT, then 100 μl of the mixtures was added to the cells and incubated at 37° C. for 2 hours. The supernatant was then removed, and the RNA was extracted using Ambion's MagMAX™-96 Total RNA Isolation Kit. All of the recovered RNA were used in a reverse transcriptase (RT) reaction with SuperScript™ III and oligo(dT) primer (Invitrogen) for synthesis of cDNA. 1 μl or 2 μl of the resulting cDNA was used for real time quantitative PCR analysis (TaqMan) using ABI Prism 7700 or 7000.
7.3.2 Results
Representative HMG1-induced cytokine release titration curves for several antibodies are shown in
Anti-HMG1 Antibodies Block HMG1 Binding to Cell Surface Receptors
Both RAGE and TLR4 have been identified as putative receptors for HMG1. To demonstrate that human anti-HMG1 antibodies are capable of blocking the interaction of HMG1 with one or more of these putative receptors, several of the human anti-HMG1 antibodies were assayed for their ability to block HMG1 binding to a RAGE-Fc fusion in an ELISA assay (
7.4.1 Materials and Methods
HMG1 binding to THP-1 Cells: Recombinant rat HMGB-1 was labeled using Eu-labelling Kit from PerkinElmer. Molar ratio of HMGB-1 to Eu is 1:5. THP-1 were cultured as the protocol from ATCC. Cells were harvested and suspended at concentration 1×106/ml in assay buffer containing 1× DELFIA L*R binding buffer (PerkinElmer), 50% of DELFIA stabilizer (PerkinElmer) and 0.05% of sodium azide. 100 μl of cells (1×105) was added into wells in 96 well cell culture plate. The plate was incubated with gentle shaking at 4° C. for one hour. 100 μl of assay buffer containing 2 nM of Europium-labeled HMGB-1 mixed with various concentrations (333, 166.5, 83.25, 41.6, 20.8, 10.4 and 5.2 nM) of human anti-HMGB1 antibodies E11 or G2 or soluble human RAGE-Fc, were added into each wells respectively. After one hour incubation with gentle shaking at 4° C., the cells were washed four times with 1× L*R wash buffer (PerkinElmer) by centrifuging at 1200 rpm×5 min. 200 μl of enhancement solution was added into each well to dissociate and enhance Eu fluorescence at 615 nm. The fluorescence was measured with Wallac Victor Fluorometer. Assay was performed in triplicate and human antibody R3-47 was used the negative isotype control.
HMG1 binding to RAGE-Ig by ELISA: 50 μl/well of RAGE-Fc fusion protein at 5 μg/ml in PBS was added to each well of an ELISA plate and incubated overnight at 4° C. The plate was then blocked with 200 μl of 5% milk at 37° C. for 1 hr and washed 3× with PBS/Tween. 50 μl/well of diluted HMGB1 solution. For dose curves, HMGB1 concentrations started at 4 ug/ml in PBS. For antibody blocking the HMGB1 was preincubated in another plate with human anti-HMG1 antibodies or buffer, then transferred to the RAGE-coated plate. The plates were then incubated at room temperature for 2 hours and washed 3×. To detect any HMG1 bound to the immobilized RAGE-Fc a mixture of biotinylated mouse anti-HMG1 InAbs (10D4, 4H11, 3E10 and 5C12) each at 1 ug/ml, total Ab: 4 ug/ml, were added to each well and the plate was incubated for 1 hour at room temperature. The plate was then washed and Strepavidin-HPR was added to each well and incubated 15 min. The plate was then washed 3× and blotted dry. 100 μl of TMB developing agent was added and the plate read at 650 nm. Values were calculated as percent of inhibition with HMG1 alone equal to 0% inhibition. The data in
TLR4 Activation Assay: HuTLR4 and CD14 stably expressed 293 cells (Invitrogen) were seeded in a 96 well plate at 2×10−4/well in 100 μl of DMEM with 10% FCS overnight. Cells were then transfected with NF-κB/Luc (Stratagene) luciferase reporter construct as indicated by the kit for 24 hr. A mixture of HMGB1 and anti-HMGB1 was added to cells at 100 μl/well overnight. The luciferase activity was then measured to reflect TLR4 activation (Promega).
7.4.2 Results
These studies demonstrate that several of the antibodies tested inhibited HMG1 binding to RAGE by at least 35% (e.g., G2, G4, S10, S16, S2 and S6), two of the antibodies tested, G2 and G4, inhibited binding by nearly 75% under the conditions tested (
Anti-HMG1 Antibodies Inhibit Sepsis in the Cecal Ligation and Puncture (CLP) Model
To demonstrate that human anti-HMG1 antibodies can inhibit lethality in sepsis, we established sepsis in mice and monitored the survival rates for several antibody treatment protocols. Mice were subjected to cecal ligation and puncture (CLP), a well characterized model of sepsis caused by perforating a surgically-created cecal diverticulum, that leads to polymicrobial peritonitis and sepsis (Fink and Heard, supra; Wichmann et al., supra; and Remick et al., supra). The survival rates for mice treated with several human anti-HMG1 antibodies was compared to mice treated with isotype control antibodies. Several human anti-HMG1 antibodies demonstrated significant protection in the CLP model including G4, S6 and S16 (
7.5.1 Materials and Methods
Anti-HMGB1 in Sepsis: To establish live intra-abdominal infection and sepsis, Balb/C mice (9-11 per group) were subjected to the CLP procedure as described previously (Fink and Heard, 1990, J Surg Res 49:186-196; Wichmann et al., 1996, J Surg Res 65, 109-114). After anesthesia with an intramuscular injection of ketamine (75 mg/kg, Fort Dodge Laboratories, Fort Dodge, Iowa) and xylazine (20 mg/kg, Boehringer Ingelheim), a 15-mm midline incision was made to expose the cecum. After ligation 5.0 mm from the tip, the cecal stump was punctured once with a 22-gauge needle, and small amount of stool (1-mm length) was extruded. The cecum was placed back into its normal intra-abdominal position, and the wound was closed with two layers of running suture. All animals received saline-solution (0.9% s.c., 20 ml/kg of body weight) resuscitation, and a single dose of antibiotic (0.5 mg of imipenem per mouse in 200 μl of sterile saline injected s.c.) (Primaxin, Merck) 30 min after surgery.
Anti-HMG1 antibodies or isotype control antibodies (50 μg/mouse in a 200 μl volume), were administered intraperitoneally at 24 and 48 hours, post surgery. In certain follow up experiments anti-HMG1 antibodies or isotype control antibodies (8 mg/kg) were administered intraperitoneally at 24 hours, post surgery (
7.5.2 Results
These studies demonstrate that the passive immunization of critically ill septic mice with human anti-HMG1 antibodies was protective. In particular the human anti-HMG1 antibodies S6, S16, E11 and G4 were protective in the CLP model (see
HMG1 is Upregulated in Animal Models of Several Inflammatory Conditions
Serum HMG1 levels have been shown to increase during sepsis/septic shock, in humans. We examined the level of HMG1 protein and/or gene expression in a number of different animal models of inflammatory disease including several arthritis models, acute lung injury and peritonitis. In all models thus far examined we found that HMG1 levels rise with disease progression. In addition, we found that the levels of several cytokines and/or putative HMG1 receptor molecules also rise.
7.6.1 Materials and Methods
Induction of inflammatory Disease: Please see below for detailed description for the methods used for the induction of each disease model. The levels of HMG1 and the various cytokines were examined in untreated animals in which disease had been induced and compared to normal animals.
HMG1 Levels in the Passive CIA Mouse: The front paws of normal or passive CIA mice were collected at day 10, snap frozen in liquid nitrogen and stored at −80° C. until assayed. Joint sample and lysis buffer was added to impact-resistant 2 ml tubes pre-filled with specialized lysing matrix A particles (Q biogene). The joint was homogenized with a FastPrep® homogenizer then centrifuged. The supernatant was collected and the HMG1 level was determined by ELISA using MesoScale technology (Meso Scale Discovery). The data shown in
Taqman Analysis of the Active CIA Mouse: The front and hind paws of normal or active CIA mice were collected at day 35, snap frozen in liquid nitrogen and stored at −80° C. until assayed. Joint sample and lysis buffer was added to impact-resistant 2 ml tubes pre-filled with specialized lysing matrix A particles (Q biogene). The joint was homogenized with a FastPrep® homogenizer and RNA was prepared from the homogenate using Qiagen's RNAse mini kit or RNA STAT-60 according to the manufacture's instructions. All of the recovered RNA was used in a reverse transcriptase reaction with SuperScript™ III and oligo(dT) primer (Invitrogen) for the synthesis of cDNA. 1 μl or 2 μl of the cDNA were used for real time quantitative PCR analysis (TaqMan) using an ABI Prism 7700 or 7000. The relative gene expression of HMGB1 and several putative receptor molecules, RAGE, TLR2, TLR4 and TLR9 were examined in both the hind and front paws separately (
HMG1 Levels in the AIA Rat: The hind paws of AIA rats were harvested at day 0, 5, 10, 15 and 20 snap frozen in liquid nitrogen and stored at −80° C. until assayed. The AIA joints were processed and assayed using the same protocol as was used for the passive CIA Mouse above. The level of HMG1 present in the joint homogenate is plotted over time in
HMG1 and Cytokine Levels in Mouse Sera After S. Aureus challenge: Serum from mice challenged with S. aureus was collected at 2, 8, and 12 hours post challenge. The levels of HMG1, IL-1b and TNF-a were determined by ELISA using MesoScale technology (Meso Scale Discovery). The levels of HMG1, IL-1b and TNF-a are plotted over time (
HMG1 Levels in the BAL fluid of the ALI Mouse: BAL fluid from mice challenged with either PBS (control) or LPS (lung injury) were harvested at the indicated times after challenge and the level of HMG1 was determined by MesoScale ELISA. The level of HMG1 is plotted over time (
HMG1 Levels in the AIA Rat Post Treatment: The hind paws of AIA rats after treatment (see Experiment 9, below) were processed as above and the levels of HMG1, IL-6 and TNF-a were determined by MesoScale ELISA.
MesoScale ELISA: Briefly, plates were pre-coated with capture antibodies for HMGB1 or cytokines (e.g., IL-1b, TNF-a, IL-6, etc) and blocked with MSD blocker buffer (MSD, Cat#R93AA-1) for 1 hour. Standards (diluted in appropriate sample buffer, for example, normal mouse BAL fluid, serum or joint homogenate) and samples were added to the plate in 20 ul volumes. 20 ul of a mixture of primary and detection antibodies were added to each well and allowed to incubate at room temperature while shaking for 4 hours. Plates were then washed, read buffer (MSD, Cat #R92TD-2) added and read on Sector Imager 6000. For HMG1 detection primary antibody was Affinity Rabbit anti-HMGB1 polyclonal antibody (Becton Dickinson Biosciences, Cat # 556528) and detection antibody was goat anti-rabbit MSD detection antibody (MSD, Cat # R32AB-1).
7.6.2 Results
Three arthritis models were examined. In each model HMG1 levels in the joint were seen to rise concurrently with disease progression. In the passive CIA mouse, the level of HMG1 increased about 10 fold by day ten (
Two other disease models were also examined.
We also examined the levels of HMG1, IL-6 and TNF-a in the joints of AIA rats after treatment with either PBS, a human isotype control (HuIgG), G4, A-box-Fc fusion and methotrexate (MTX) in conbination with either HuIgG or Renbrel or G4.
Anti-HMG1 Antibodies Inhibit the Severity of Disease Progression in the Passive Collagen-Induced Arthritis (CIA) Mouse Model
To demonstrate that a human antibody against HMG1 was a useful therapeutic we tested a panel of human anti-HMG1 antibodies to treat collagen-induced arthritis in a passive mouse model. For this series of experiments we utilized a prevention model in which treatment is initiated prior to the onset of clinical arthritis. In this study we directly compared the efficacy of anti-HMG1 antibodies with that of known treatment protocols, either Renbrel (an accepted Enbrel™ surrogate molecule for use in rodent model systems) alone or the combination of Renbrel™ and methyltrexate (MTX).
We demonstrate here, for the first time, that an antibody against HMG1 demonstrated efficacy in a prevention in a passive CIA mouse RA model. In fact, the anti-HMG1 antibody G4 was shown to be more effective then Renbrel alone while the anti-HMG1 antibody S6 was more effective then even MTX/Renbrel therapy in reducing paw inflammation and in reducing bone loss and cartilage damage.
7.7.1 Materials and Methods
Induction of Passive Collagen Induced Arthritis (CIA): To establish an arthritis model six-eight week old male DBA/1J mice (Jackson Labs, Bar Harbor, Me.) were used. Generally 5-8 mice per group are used, On day 0, mice were immunized with 2 mg/mouse of anti-collage mAb cocktail (Chemicon # ECM 1100, 10 mg/ml) intravenously, i.v. at tail. Mice were subsequently injected with 50 μg LPS/mouse, i.p. on day 3. Each experiment had several groups of animals as follows: groups A-E in experiment 1, groups G-J in experiment 2 and groups L-N). An additional group of mice (groups F, K and O) were untreated as normal controls. The following treatments were administered as shown in Table 4.
Monitoring Disease: Beginning at day 0 all animals were observed daily to assess the status of the disease in their paws, which was done by assigning a qualitative clinical score to each of the paws. Every day, each animal has its 4 paws scored according to its state of clinical disease. The scoring is done by two observers, with at least one blinded. In addition, each mouse is weighed, to follow body weight changes, at the same time the paws are scored (see
Grading scales for the ankle/wrist/midfoot/forfoot are as follows:
Histology: Hind-limb tibiotalus joints from each animal were evaluated and scored for histologic changes as described in Badger et al., 2001, Arthritis & Rheumatism 44:128-37. Briefly, animals were sacrificed on day 32 and the hind legs were fixed in formaline and decalcified in Cal-Rite (Richard-Allen Scientific, Kalamazoo, Mich.). The paws were then removed from the legs at the distal tibial diaphysis. After routine processing, the samples were embedded and coronal sections were cut in the plane midway through the tibiotalus and talartarsal joints. Sections were stained with Safranin O and counterstained with fast Green (data not shown).
The histological characteristic of the bone and articular cartilage/periarticular soft tissue were scored separately by a blinded observer (
7.7.2 Results
Several experiments were performed to demonstrate that treatment with HMG1 antibodies could prevent or reduce disease severity in the passive CIA mouse model. In experiment 1 mice were treated with anti-HMG1 antibody S6 or G16 (0.2 mg/mouse) starting on day 3 after mice had been injected with LPS. Mice received a total of 6 doses over a thirteen day period. At the same time control mice received either human mAb (0.2 mg/mouse). A final group received a combination therapy of MTX (0.2 mg/mouse, 4 doses over a twelve day period) and Renbrel (0.2 mg/mouse, 2 doses over a ten day period). The development of arthritis was assessed daily after the initial treatment. The graphs in
Another clinical feature of disease progression is weight loss. The relative body weight scores for the control animal show a net decrease during the course of the study. Although the clinical scores for the mice treated with anti-HMG1 S6 antibody showed significant protection, this group of animals also showed a net decrease in bodyweight. However the anti-HMG1 S6 antibody treated animals did not lose as much as the control group. The MTX/Renbrel treated animals also showed a net decrease in bodyweight early in the study however they had caught up with untreated animals by the end (
In experiments 2 and 3 mice were treated with the anti-HMG1 antibody G4 (10 mg/kg) starting on day 3 after mice had been injected with LPS. Mice received a total of 6 doses over a thirteen day period in experiment 2 and a total of 4 doses over the same time period in experiment 3. At the same time control mice received either human mAb (10 mg/kg) or PBS. In experiment 2 final group received Renbrel (dosing was the same as for G4). The development of arthritis was assessed daily after the initial treatment. The graphs in
Anti-HMG1 Antibodies Inhibit the Severity of Disease Progression in the Active Collagen-Induced Arthritis (CIA) Mouse Model
To demonstrate that a human antibody against HMG1 was a useful therapeutic we tested a panel of anti-HMG1 antibodies to treat active collagen-induced arthritis in a mouse model. For this series of experiments we utilized a prevention model in which treatment is initiated prior to the onset of clinical arthritis. In this study we compared the efficacy of anti-HMG1 antibodies with that of Renbrel.
We demonstrate here, for the first time, that an antibody against HMG1 demonstrated efficacy in reducing paw inflammation and weight loss in an active CIA mouse RA model. In fact, the anti-HMG1 antibody G4 was shown to be more effective then Renbrel alone in reducing paw inflammation.
7.8.1 Materials and Methods
Induction of Active Collagen Induced Arthritis (CL4): Six-eight week old male DBA/1J mice (Jackson Labs, Bar Harbor, Me.) were used. On day 0, isoflurane anesthetized animals were given an intradermal injection, at base of tail, of 200 μg bovine Type II collagen (CII) dissolved in 50 μl 0.1 N acetic acid and emulsified with an equal volume of complete Freund's adjuvant (Chondrex, Redmond, Wash.). Three weeks later on day 21 they were given a second similar intradermal injection of 100 μg of CII dissolved in 25 μl 0.1N acetic acid and emulsified with an equal volume of incomplete Freund's adjuvant (Difco, Detroit, Mich.).
Monitoring Disease: Beginning at day 14 all animals were observed daily to assess the status of the disease in their paws, which was done by assigning a qualitative clinical score to each of the paws. Every day, each animal has its 4 paws scored according to its state of clinical disease. The scoring is done by two observers, with at least one blinded. In addition, each mouse is weighed, to follow body weight changes, at the same time the paws are scored.
Grading scales for the ankle/wrist/midfoot/forfoot are as follows:
We examined whether treatment with human anti-HMG1 antibodies could prevent or reduce disease severity CIA in an active CIA model. Mice were treated with either anti-HMG1 antibody G4, an isotype control antibody or Renbrel at 10 mg/kg, every three days starting on day 21. The development of arthritis was assessed daily. The graphs in
Another clinical feature of disease progression in this model is weight loss. The relative body weight scores for the control animal show a net decrease during the course of the study. Although the clinical scores for the mice treated with anti-HMG1 G4 antibody showed significant protection, this group of animals also showed a net decrease in bodyweight. However the anti-HMG1 G4 antibody treated animals did not lose as much as the control group. These results demonstrate that anti-HMG1 antibodies can effectively protect against joint damage and other symptoms when administered prior to the onset of disease. In particular, these results indicate that anti-HMG1 G4 antibody treatment had a profound effect in diminishing the disease severity in the active CIA mouse model.
Anti-HMG1 Antibodies Inhibit the Severity of Disease Progression in the Adjuvant-Induced Arthritis (AIA) Rat Model
We further tested the human anti-HMG1 antibody G4 in the Adjuvant-Induced Arthritis (AIA) Rat Model. For this series of experiments we utilized a prevention model in which treatment is initiated prior to the onset of clinical arthritis. In this study we compared the efficacy of anti-HMG1 antibodies with that of Renbrel.
We demonstrate here, for the first time, that an antibody against HMG1 (G4) demonstrated efficacy in reducing paw inflammation in the AIA Rat RA model. In fact, the anti-HMG1 antibody G4 was shown to be more effective then Renbrel alone in reducing paw inflammation. An approximately 34% reduction in clinical scores was seen for the anti-HMG1 G4 animals while only an approximately 11% reduction was seen for the Renbrel treated animals. In addition we demonstrate that the combinantion of methotrexate and G4 was more effective at reducing paw inflammation scores than a combinantion of methotrexate and Renbrel.
7.9.1 Materials and Methods
Induction of Adjuvant Induced Arthritis (AIA): Six-eight week old female DA rats (Harlen) were used. On day 0, isoflurane anesthetized animals were given an intradermal injection, at base of tail, 0.75 mg of mycobacterium butyricum (Difco #0640-33-7) mixed in 100 μl incomplete Freund's adjuvant (Difco, Detroit, Mich.). The following treatments were administered as shown in Table 5.
The human anti-HMG1 antibody G4, the A-box-Fc fusion and the huIgG isotype control were administered at 10 mg/kg, every 3 days, day 0-15. Methotrexate was administered at 0.8 mg/kg, every 6 days, day 0-15 and Renbrel was administered at either 2.5 mg/kg every two days, day 0-15 for treatment group 5 or at 4 mg/kg, every 3days, day 0-15 for treatment group 6.
Monitoring Disease: Beginning at day 6 all animals were observed daily to assess the status of the disease in their paws, which was done by assigning a qualitative clinical score to each of the paws. Every day, each animal has its 4 paws scored according to its state of clinical disease. The scoring is done by two observers, with at least one blinded. In addition, each mouse is weighed, to follow body weight changes, at the same time the paws are scored.
Grading scales for the ankle/wrist/midfoot/forfoot are as follows:
The joints of the 4 lateral digits of each paw are graded as involved or not involved, i.e., 1 or 0. For example, a maximally involved left rear paw would be scored: ankles=4, midfoot=4, MTP=4, PIP=4, DIP=4 (20 units). We would repeat this for each paw and sum the scores. Rats are euthanized at day 21, or sooner if total clinical score reaches 80 and a histological evaluation of joints is performed.
7.9.2 Results
We determined that the human anti-HMG1 antibody G4 could prevent or reduce the severity of AIA in rats. AIA rats were treated with either PBS, anti-HMG1 antibody G4, an isotype control antibody (HuIgG) at 10 mg/kg, every three days or Renbrel at 2.5 mg./kg, every two days starting on day 21. In addition, AIA rats were treated with methotrexate in combination with several other therapies including Renbrel, G4 or HuIgG. AIA rats were also treated with an HMG1 A-box-Fc fusion protein. The development of arthritis was assessed daily. The graphs in
The graphs in
Anti-HMG1 Antibodies Improve Survival in the S. aureus Mouse Model of Peritonitis
We also tested several human anti-HMG1 antibody in a severe mouse model of peritonitis where the serum levels of HMG1 were seen to rise (see above). Here we demonstrate that treatment with a human anti-HMG1 antibody improves survival by nearly 30% over controls.
7.10.1 Materials and Methods
Induction of Peritonitis: Four-six week old female BALB/c mice were used. An initial study with mice challenged i.p. with heat inactivated S. aureus premixed with galactosamine demonstrated that the LD100 was between 1×107 and 1×109 cells (data not shown). For determination of HMG1 levels on day 0 approximately 109 heat inactivated S. aureus (strain 8325-4) cells premixed with 20 mg of galactosamine or galactosamine alone in a 200 μl volume of PBS was administered i.p. A third group of animals was not challenged. Animals were euthanized at 2, 8, and 12 hours after challenge by CO2 inhalation and blood was collected by cardiac puncture. For the antibody treatment studies on day 0 approximately 109 heat inactivated S. aureus (strain 8325-4) cells premixed with 20 mg of galactosamine were administered i.p. in a 200 μl volume and the following treatments were administered i.p. in a 100 μl volume 30 minutes prior to challenge as shown in Table 6.
S. aureus
S. aureus
S. aureus
S. aureus
Monitoring Disease: Starting on day 1 and continuing through day 14 the animals were observed daily for signs of morbidity (severely decreased mobility, ruffled fur, and/or loss of >20% of highest body weight) and mortality. The animals were also weighed 2× weekly. Animals showing signs of significant morbidity were euthanized. On day 15 all surviving animals were euthanized (euthanized sooner if significant morbidity observed).
7.10.2 Results
Human anti-HMG1 antibodies were also tested in a severe gram-positive bacterium induced septicemia model in which mice were challenged with a lethal dose of heat inactivated S. aureus. In this model none of the mice treated with either PBS or the antibody isotype control (R347) survived. In contrast 27% of the mice treated with the human antibody against HMG1 G4 survived (
Anti-HMG1 Antibodies Reduce Cellular Infiltration Associated with Acute Lung Injury
Two human anti-HMG1 antibodies, were tested in the lipopolysaccharide (LPS) induced acute lung injury (ALI) mouse model. Here we demonstrate for the first time that treatment with a human anti-HMG1 antibody reduces total cellular infiltration by approximately 40% compared to controls (
7.11.1 Materials and Methods
Induction of LPS-induced ALI: LPS (E. coli strain 0111:B4 . . . Sigma, St. Louis, Mo.) was administered intranasally to isoflurane (Baxter Pharmaceuticals, Deerfield, Ill.) anesthetized adult BALB/c mice on day 1 at a dose of 5-10 ug per mouse in 50-100 μl. For determination of HMG1 levels animals were euthanized by CO2 asphyxiation at 4, 8, 24, 32 and 48 hours post LPS administration, and BronchoAlveolar Lavarge (BAL) and other samples collected for protein analysis and histopathology. For treatment protocols 24 hours post LPS dosing, anti-HMGB1 antibodies, HMG1 A-box Fc fusion or controls were administered intraperitoneally (i.p.) at a dose of 10 mg/kg in 100ul volumes. On day 3 (48 h post LPS dosing), animals were euthanized by CO2 asphyxiation and samples (BALs, blood, and lungs) collected for analysis.
BAL Sample Collection: lungs were flushed three times with ˜0.8 ml Phosphate buffered saline (PBS, pH 7.2, GIBCO, Rockville, Md.) using a syringe with a catheter tubing. BAL samples collected were centrifuged at 1,200 rpm for 10 min at 4C, supernatant collected and stored at −80C for protein (eg. HMGB1) quantitation, and cells in pellet were resuspended and transferred to cytoslides, fixed, GIEMSA stained, and BAL cellularity determined visually with the aid of a microscope.
7.11.2 Results
Human anti-HMG1 antibodies were tested in an acute lung injury model induced by intranasal administration of LPS. In this model the HMG1 A-box, a known competitive inhibitor of HMG proinflammatory action, only reduced infiltration by about 23% compared to controls. In contrast G4 and E11 were seen to reduce the total cellular infiltrate present in the BAL fluid by 37%-40% compared to controls, demonstrating that anti-HMG1 antibodies are useful for the treatment of acute lung injury.
HMGB1 Staining Patterns in Multiple Sclerosis (MS) Plaques
HMGB staining patterns were examined in MS plaques from human brain tissue using the G16 human anti-HMGB antibody. Plaques with demyelination and numerous activated microglia as well as plaques with predominantly demyelination, few activated microglia and numerous lymphocytes were examined.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.
This application claims the benefit under 35 U.S.C. §119(e) of the following U.S. Provisional Application Nos.: 60/620,726 filed Oct. 22, 2004; 60/651,512 filed Feb. 10, 2005; 60/658,572 filed Mar. 7, 2005; 60/662,944 filed Mar. 18, 2005 and 60/713,712 filed Sep. 6, 2005. The priority applications are hereby incorporated by reference herein in their entirety for all purposes.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5545806 | Lonberg et al. | Aug 1996 | A |
| 5545807 | Surani et al. | Aug 1996 | A |
| 5594114 | Goodearl et al. | Jan 1997 | A |
| 5605690 | Jacobs et al. | Feb 1997 | A |
| 5656272 | Le et al. | Aug 1997 | A |
| 5859205 | Adair et al. | Jan 1999 | A |
| 6177077 | Tobinick | Jan 2001 | B1 |
| 6303321 | Tracey et al. | Oct 2001 | B1 |
| 6323329 | Bullerdiek | Nov 2001 | B1 |
| 6448223 | Tracey et al. | Sep 2002 | B1 |
| 6468533 | Tracey et al. | Oct 2002 | B1 |
| 7304034 | Tracey et al. | Dec 2007 | B2 |
| 20020009749 | Ozaki et al. | Jan 2002 | A1 |
| 20030017155 | Tracey et al. | Jan 2003 | A1 |
| 20030060410 | Tracey et al. | Mar 2003 | A1 |
| 20030113323 | Tracey et al. | Jun 2003 | A1 |
| 20030143194 | Tracey et al. | Jul 2003 | A1 |
| 20040053841 | Tracey et al. | Mar 2004 | A1 |
| 20040120953 | Tracey et al. | Jun 2004 | A1 |
| 20040141948 | O'Keefe | Jul 2004 | A1 |
| 20050118688 | Freeze et al. | Jun 2005 | A1 |
| 20050152903 | Newman et al. | Jul 2005 | A1 |
| 20060057679 | O'Keefe et al. | Mar 2006 | A1 |
| 20060099207 | Wu et al. | May 2006 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 079 849 | Jan 2002 | EP |
| 62-166897 | Jul 1987 | JP |
| 2003-096099 | Apr 2003 | JP |
| WO-96-25493 | Aug 1996 | WO |
| WO-97-23611 | Jul 1997 | WO |
| WO-99-59609 | Nov 1999 | WO |
| WO-00-47104 | Aug 2000 | WO |
| WO-02-074337 | Sep 2002 | WO |
| WO-02-092004 | Nov 2002 | WO |
| WO-02-092004 | Nov 2002 | WO |
| WO-2004-004763 | Jan 2004 | WO |
| WO-2005-034952 | Apr 2005 | WO |
| WO-2007-054090 | May 2007 | WO |
| WO-2007-076200 | Jul 2007 | WO |
| WO-2007-084253 | Jul 2007 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060099207 A1 | May 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60713712 | Sep 2005 | US | |
| 60662944 | Mar 2005 | US | |
| 60658572 | Mar 2005 | US | |
| 60651512 | Feb 2005 | US | |
| 60620726 | Oct 2004 | US |
