JP2006510619A - Use of HMGB fragments as anti-inflammatory agents - Google Patents

Abstract

Disclosed are compositions and methods for inhibiting the release of pro-inflammatory cytokines from cells and for inhibiting the inflammatory cytokine cascade in patients. The composition comprises an antibody preparation that specifically binds to the HMGB A box, and / or HMGB B box, and / or an inhibitor of TNF biological activity. The method includes treating the cell or patient with an amount of the composition sufficient to inhibit the release of pro-inflammatory cytokines or to inhibit the inflammatory cytokine cascade.

Description

Detailed Description of the Invention

Related Applications This application claims the benefit of US patent applications 60 / 427,841 and 60 / 427,846, both filed on Nov. 20, 2002. The entire contents of both applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION Inflammation is often a pro-inflammatory cytokine such as tumor necrosis factor (TNF), interleukin (IL) -1α, IL-1β, IL-6, platelet activating factor (PAF), macrophage migration inhibitory factor (MIF) and Often induced by other compounds. These pro-inflammatory cytokines are of several different cell types, most importantly not only by immune cells (eg monocytes, macrophages and neutrophils) but also non-immune cells such as fibroblasts, osteoblasts, smooth It is also produced by muscle cells, epithelial cells and neurons. These pro-inflammatory cytokines contribute to various diseases in the early stages of the inflammatory cytokine cascade.

  The inflammatory cytokine cascade contributes to deleterious features including inflammation and apoptosis in many diseases. These include disorders characterized by both local and systemic reactions, such as diseases involving the gastrointestinal tract and related tissues (eg appendicitis, peptic ulcer, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcers) Sex, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottis, anorexia, cholangitis, cholecystitis, celiac disease, hepatitis, Crohn's disease, enteritis and whipple disease); systemic or local inflammation Sex diseases and conditions (eg, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, abnormally high fever, favorable Eosinophilic granulomas, granulomatosis and sarcoidosis); diseases involving the urological system and related tissues (eg septic abortion, epididymis, vaginitis, prostatitis and Urethritis); diseases involving the respiratory system and related tissues (eg bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonia, adult respiratory distress syndrome, pulmonary ultramicroscopic silicon voluntary pneumonia, alveolitis, bronchiole Flames, laryngitis, pleurisy and sinusitis); various viruses (eg influenza, respiratory syncytium virus, HIV, hepatitis B virus, hepatitis C virus and herpes, bacteria (eg disseminated bacteremia, dengue fever), Diseases resulting from infection with mold (eg candidiasis) and protozoa and multicellular parasites (eg malariasis, filariasis, amebiasis and hepatic cystosis); dermatological diseases and skin conditions (eg burns, dermatitis, skin Myositis, sunburn, urticaria and wheal; diseases involving the cardiovascular system and related tissues (eg vasculitis, vasculitis, endocarditis, arteritis, atherosclerosis) Venous sclerosis, restenosis, thrombophlebitis, epicarditis, congestive heart failure, myocarditis, myocardial ischemia, nodular periarteritis and rheumatic fever); central and peripheral nervous system and related tissues involved Diseases (eg Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Gann-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis and uveitis); bones, joints, muscles and connections Diseases of tissues (eg various arthritis and joint pain, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis and periosteitis); other autoimmune and inflammatory diseases (eg myasthenia gravis) , Thyroiditis, systemic lupus erythematosus, Goodpasture syndrome, Behcet syndrome, allograft rejection, graft-versus-host disease, type I diabetes, ankylosing spondylitis, Buerger's disease and Reiter syndrome) And various cancers, tumors and proliferative disorders (eg, Hodgkin's disease); and in any case, but not limited to, inflammatory or immune host responses to any primary disease.

  Early pro-inflammatory cytokines (eg TNF, IL-1 etc.) mediate inflammation and accumulate in the high mobility group box 1 (HMGB1) (also known as HMG-1 and HMG1), ie serum Induces late release of proteins that mediate further induction of delayed lethal and early pro-inflammatory cytokines.

  HMGB1 was first discovered as a fundamental member of a family of DNA-binding proteins called high mobility group box (HMGB) proteins that are important for DNA structure and stability. It was discovered about 40 years ago as a ubiquitously expressed nuclear protein that binds to double-stranded DNA without sequence specificity.

  HMGB1 binding promotes DNA formation and stability of nucleoprotein complexes that facilitate gene transcription of the glucocorticoid receptor and RAG recombinase. The HMGB1 molecule has three domains, two DNA-binding motifs called HMGB A and HMGB B boxes and an acidic carboxyl terminus. The two HMGB boxes are highly conserved 80 amino acid L-type domains. The HMGB box is also expressed in other transcription factors including RNA polymerase I transcription factor, human upstream binding factor and lymphocyte-like specific factor.

  Recent evidence suggests that HMGB1 is an inflammatory cytokine mediator. For example, HMGB1 is regarded as a delayed lethal cytokine mediator in endotoxemia. According to this study, bacterial endotoxin (lipopolysaccharide (LPS)) activates monocytes / macrophages and releases HMGB1 as a late response to activation, resulting in an elevated plasma HMGB1 concentration that is toxic ing. Antibodies against HMGB1 prevent endotoxin lethality even if antibody administration is delayed until after the initial cytokine response. Like other proinflammatory cytokines, HMGB1 is a potent activator of monocytes. Intratracheal administration of HMGB1 leads to acute lung injury, and anti-HMGB1 antibody shows a protective action against endotoxin-induced lung edema. Serum HMGB1 levels are elevated in clinically afflicted patients with sepsis or hemorrhagic shock and levels are significantly higher in non-survivors compared to survivors.

  HMGB1 has also been designated as a ligand for RAGE, the multiligand receptor of the immunoglobulin superfamily. RAGE is expressed in endothelial cells, smooth muscle, monocytes and nerves, and ligand interactions signal through MAP kinase, P21ras and NF-κB. The delayed kinetics of HMGB1 appearance during endotoxemia make it a potentially good therapeutic agent, but little is known about the molecular mechanisms of HMGB1 signaling and toxicity.

  Thus, it is useful to discover the characteristics of HMGB1 pro-inflammatory activity, particularly the inhibitory action of either the active domain or other domains that are responsible for this activity.

SUMMARY OF THE INVENTION The present invention provides that (1) HMGB A box functions as a competitive inhibitor of HMGB pro-inflammatory action, (2) HMGB B box has HMGB's dominant pro-inflammatory activity, and (3) HMGB Based on the discovery that combined therapies that include agents that inhibit biological activity and agents that inhibit the biological activity of TNF can be used for the treatment of conditions characterized by activation of the inflammatory cytokine cascade. Agents that inhibit the biological activity of HMGB include the HMGB A box, which functions as a competitive inhibitor of HMGB's pro-inflammatory action and an antibody against HMGB, such as the HMGB B box.

  Accordingly, in one embodiment, the present invention provides a high mobility group box protein (HMGB) A box or a thereof that can inhibit the release of proinflammatory cytokines from cells administered a high mobility group box (HMGB) protein. A variant, or a polypeptide containing an A-box biologically active fragment or variant thereof, where HMGB A-box is HMG1L5 (formerly HMG1L10) A-box, HMG1L1A-box, HMG1L4A-box, BAC clone RP11- Selected from the group consisting of 395A23 HMGB A box polypeptide, HMG1L9A box, LOC122441A box, LOC139603A box and HMG1L8A box. In one embodiment, the polypeptide can be present in a pharmaceutically acceptable carrier.

  In another embodiment, the invention is a purified preparation of an antibody that specifically binds to a high mobility group box protein (HMGB) B box but does not specifically bind to a non-B box epitope of HMGB; Here the antibody can suppress the release of pro-inflammatory cytokines from cells administered HMGB, and HMGB B box is HMG1L5 (formerly HMG1L10) B box, HMG1L1B box, HMG1L4B box, BAC clone RP11-395A23 Selected from the group consisting of HMGB B box polypeptides. In one embodiment, the antibody can be present in a pharmaceutically acceptable carrier.

  In yet another embodiment, the present invention relates to a high mobility group box protein (HMGB) B box or variant thereof, or a B box biologically active fragment or variant thereof, but not a full-length HMGB. Is a peptide, where the polypeptide can induce the release of pro-inflammatory cytokines from cells, and the HMGB B box is a HMG1L5 (formerly HMG1L10) B box, HMG1L1B box, HMG1L4B box, BAC clone RP11-395A23 Selected from the group consisting of HMGB B box polypeptides. In one embodiment, the polypeptide can be present in a pharmaceutically acceptable carrier.

  In another embodiment, the present invention includes a vector encoding the above-described polypeptide.

  In yet another embodiment, the invention is a method of inhibiting the release of pro-inflammatory cytokines from mammalian cells, wherein the method specifically binds to a high mobility group box protein (HMGB) B box but is non-HMGB. It involves administering to a cell an amount of a purified preparation of an antibody that does not specifically bind to the B box epitope, where HMGB B box is HMG1L5 (formerly HMG1L10) B box, HMG1L1B box, HMG1L4B box , Selected from the group consisting of HMGB B box polypeptides of BAC clone RP11-395A23.

  In another embodiment, the invention is a method of inhibiting the release of proinflammatory cytokines from mammalian cells, the method comprising a high mobility group in an amount sufficient to inhibit the release of proinflammatory cytokines from the cells. High mobility group box protein (HMGB) A box or variant thereof, or biologically active fragment of A box or variant thereof, capable of suppressing the release of pro-inflammatory cytokines from cells administered box (HMGB) protein MGGB A box includes HMG1L5 (formerly HMG1L10) A box, HMG1L1A box, HMG1L4A box, BAC clone RP11-395A23 HMGB A box polypeptide, HMG1L9A box , LOC122441A box, LOC139603A box, and HMG1L8A box. In one embodiment, the cells can be administered a vector encoding a polypeptide comprising an A box polypeptide, a biologically active fragment of an A box, or a variant thereof.

  In another embodiment, the invention is a method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade, comprising an amount of a high mobility group box protein sufficient to inhibit the inflammatory cytokine cascade. (HMGB) comprising administering to a patient a purified preparation of an antibody that specifically binds to a B box but not specifically to a non-B box epitope of HMGB, wherein the HMGB B box is HMG1L5 (formerly HMG1L10) B box, HMG1L1B box, HMG1L4B box, BAC clone RP11-395A23 selected from the group consisting of HMGB B box polypeptides.

  In another embodiment, the invention is a method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade, comprising an amount sufficient to inhibit the release of proinflammatory cytokines from the cells, High mobility group box protein (HMGB) A box or variant thereof, or biologically active fragment of A box, which can suppress the release of pro-inflammatory cytokines from cells administered high mobility group box (HMGB) protein Or administration of a polypeptide comprising a variant thereof to a patient, wherein the HMGB A box is HMG1L5 (formerly HMG1L10) A box, HMG1L1A box, HMG1L4A box, HMGB A box polypeptide of BAC clone RP11-395A23 , HMG1L9A box, LOC122441B box, LOC139603A box and HMG1L8A box.

  In yet another embodiment, the present invention provides a high mobility group box protein (HMGB) B box or variant thereof, or a B box organism, in an amount sufficient to promote the release of proinflammatory cytokines from the cell. Is a method of promoting the release of proinflammatory cytokines from a cell comprising administering to the cell a polypeptide comprising a biologically active fragment or variant thereof but not full length HMGB, wherein the HMGB B box is HMG1L5 ( Previous HMG1L10) B box, HMG1L1B box, HMG1L4B box, selected from the group consisting of HMGB B box polypeptides of BAC clone RP11-395A23. In one embodiment, the cells can be administered a vector encoding a polypeptide comprising a B box polypeptide, a biologically active fragment of a B box, or a variant thereof.

  In yet another embodiment, the invention is a method for causing weight loss or treating obesity in a patient, which comprises an amount sufficient to promote the release of pro-inflammatory cytokines from cells. High mobility group box protein (HMGB) B box or variant thereof, or an effective amount of a polypeptide containing a B box biologically active fragment or variant thereof, but not full length HMGB polypeptide Wherein the HMGB B box is selected from the group consisting of HMG1L5 (formerly HMG1L10) B box, HMG1L1B box, HMG1L4B box, HMGB B box polypeptide of BAC clone RP11-395A23.

  In another embodiment, the present invention is a method for determining whether a compound inhibits inflammation, comprising: a) high mobility group box protein (HMGB) B box or biologically Cells that release pro-inflammatory cytokines when exposed to active fragments thereof; and b) in combination with an HMGB B box or biologically active fragment thereof, wherein the HMGB B box is HMG1L5 (formerly HMG1L10 ) It is selected from the group consisting of B box, HMG1L1B box, HMG1L4B box, HMGB B box polypeptide of BAC clone RP11-395A23; next, the compound suppresses the release of pro-inflammatory cytokines from cells Whether or not.

  In yet another embodiment, the present invention provides a high mobility group box capable of inhibiting the release of proinflammatory cytokines from cells administered a high mobility group box (HMGB) protein in a pharmaceutically acceptable carrier. A protein (HMGB) A box or fragment or variant thereof and an agent that inhibits the biological activity of TNF, wherein the agent is selected from the group consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide Is a pharmaceutical composition comprising a polypeptide comprising. The HMGB A box is preferably a vertebrate HMGB A box, such as a mammalian HMGB A box, more preferably a mammalian HMGB1 A box, such as a human HMGB1 A box, most preferably SEQ ID NO: 4, SEQ ID NO: 22 or SEQ ID NO: 57. HMGB1 A box containing or consisting of sequences.

  In another embodiment, the present invention binds to an HMGB polypeptide or biologically active fragment thereof, such as an HMGB B box polypeptide or biologically active fragment thereof, in a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody and an agent that inhibits the biological activity of TNF, wherein the agent is selected from the group consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide .

  In yet another embodiment, the present invention relates to a high mobility group box protein (HMGB) A box or its, which can inhibit the release of pro-inflammatory cytokines from cells administered a high mobility group box (HMGB) protein. A polypeptide comprising a fragment or variant, and an agent that inhibits the biological activity of TNF, wherein the agent is selected from the group consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide; A method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade comprising administering to the patient a composition comprising:

  In yet another embodiment, the invention provides an antibody that binds to an HMGB polypeptide or biologically active fragment thereof, eg, an HMGB B box polypeptide or biologically active fragment thereof, and a TNF organism. Inflammation, comprising administering to a patient a composition comprising an agent that inhibits pharmacological activity, wherein the agent is selected from the group consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide A method of treating a condition in a patient characterized by activation of a cytokine cascade.

Detailed Description of the Invention Unless otherwise stated, the practice of the present invention employs conventional techniques of cell culture, molecular biology, microbiology, cell biology and immunology well known to those skilled in the art. Such techniques are explained fully in the literature. For example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995), “Short Protocols in Molecular Biology”, John Wiley and Sons; Methods in Enzymology (several volumes) ); Methods in Cell Biology (several volumes) and Methods in Molecular Biology (several volumes).

  The present invention is based on a series of discoveries that further elucidate the various properties of HMGB1's ability to induce pro-inflammatory cytokine production and the inflammatory cytokine cascade. In particular, the pro-inflammatory active domain of HMGB1 is the B box (especially the first 20 amino acids of the B box), and antibodies specific to the B box inhibit pro-inflammatory cytokine release and the inflammatory cytokine cascade, resulting in inflammatory cytokines It was discovered that adverse symptoms induced by the cascade can be reduced. Furthermore, the A box was a weak agonist of inflammatory cytokine release and competitively suppressed the pro-inflammatory activity of the HMGB1 B box. Furthermore, a pharmaceutical composition for use in the treatment of a condition characterized by activation of an inflammatory cytokine cascade in a patient by combining an inhibitor of biological activity of TNF with an HMGB A box and / or HMGB1 antibody. It was found that it can be formed.

  As used herein, an “HMGB polypeptide” or “HMGB protein” is a substantially pure or substantially pure and isolated polypeptide, which is a component that naturally accompanies it. Or a synthetic or recombinantly produced polypeptide having the same amino acid sequence and increases inflammation and / or increases the release of proinflammatory cytokines from cells and / or Increases the activity of the inflammatory cytokine cascade. In one embodiment, the HMGB polypeptide has one of the biological activities described above. In another embodiment, the HMGB polypeptide has two of the biological activities described above. In a third embodiment, the HMGB polypeptide has all three of the biological activities described above.

  Preferably, the HMGB polypeptide is a mammalian HMGB polypeptide, such as a human HMGB1 polypeptide. Examples of HMGB polypeptides include polypeptides comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 18. Preferably, the HMGB polypeptide contains a B box DNA binding domain and / or an A box DNA binding domain, and / or an acidic carboxyl terminus, as described herein. Other examples of HMGB polypeptides are described in GenBank accession numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP_002119, CAA31110, S02826, U00431, X67668, NP_005333, NM_016957 and J04179, which are incorporated by reference in their entirety. Are incorporated herein. Other examples of HMGB polypeptides include, for example, mammalian HMG1 (eg, described in GenBank accession number U51677 (HMGB1)), HMG2 (eg, described in GenBank accession number M83665 (HMGB2)), HMG-2A (eg, GenBank accession numbers NM_005342 and NP_005333 (HMGB3, HMG-4)), HMG14 (for example, those described in GenBank accession number P05114), HMG17 (for example, those described in GenBank accession number X13546), HMGI (eg as described in GenBank accession number L17131), and HMGY (eg as described in GenBank accession number M23618); non-mammalian HMGT1 (eg as described in GenBank accession number X02666) and HMGT2 ( For example, those described in GenBank accession number L32859) (rainbow trout); HMG-X (for example, described in GenBank accession number D30765) (Xenopus laevis), HMGD (eg GenBank accession number X71138) and HMGZ (eg GenBank accession number X71139) (Drosophila); NHP10 protein (HMG protein homolog NHP1) (eg GenBank accession number Z48008) Non-histone chromosomal proteins (such as those described in GenBank accession number O00476) (kobo); HMG1 / 2-like proteins (such as those described in GenBank accession number Z11540) (wheat, corn, Soybean); upstream binding factor (UBF-1) (for example, described in GenBank accession number X53390); PMS1 protein homolog 1 (for example, described in GenBank accession number U13695); single chain recognition protein (SSRP) , Structure-specific recognition proteins) (eg those described in GenBank accession number M86737); HMG homolog TDP- 1 (for example, as described in GenBank accession number M74017); mammalian sex determination region Y protein (SRY, testis determinant) (for example, as described in GenBank accession number X53772); mold protein: mat-1 (for example GenBank accession number AB009451), ste11 (e.g. described in GenBank accession number X53431) and Mc1; SOX14 (e.g. described in GenBank accession number AF107043) and SOX1 (e.g. GenBank accession number) Y13436), SOX2 (for example, described in GenBank accession number Z31560), SOX3 (for example, described in GenBank accession number X71135), SOX6 (for example, described in GenBank accession number AF309034) SOX8 (for example, the one described in GenBank accession number AF226675), SOX10 (for example, the one described in GenBank accession number AJ001183), SOX1 2 (eg as described in GenBank accession number X73039) and SOX21 (eg as described in GenBank accession number AF107044); lymphoid specific factor (LEF-1) (eg as described in GenBank accession number X58636) T cell-specific transcription factor (TCF-1) (eg, as described in GenBank accession number X59869); MTT1 (eg, as described in GenBank accession number M62810); and SP100-HMG nuclear self Including, but not limited to, antigens (eg, those described in GenBank accession number U36501).

  Other examples of HMGB proteins are GenBank accession numbers NG_000897 (and especially nucleotides 150-797 of NG_000897 described in FIGS. 14A and 14B); AF076674 (HMG1L1) (and especially the nucleotides of AF076674 described in FIGS. 14C and 14D) 1-633); AF076676 (HMG1L4) (and especially nucleotides 1-564 of AF076676 described in FIGS. 14E and 14F); AC010149 (HMG sequence from BAC clone RP11-395A23) (and specifically described in FIGS. 14G and 14H) AF165168 (HMG1L9) (and especially AF165168 nucleotides 729-968 described in FIGS. 14I and 14J); XM — 063129 (LOC122441) (and specifically described in FIGS. 14K and 14L) XM_063129 nucleotides 319-558); XM_066789 (LOC139603) (and nucleotides 1-258 of XM_066789 specifically described in FIGS. 14M and 14N); and AF165167 (HMG1L8) In particular a polypeptide encoded by HMGB nucleic acid sequence having nucleotides 456-666) of AF165167 as described (HMG1L5 (previous HMG1L10)) in FIG. 14O and 14P.

  The HMGB polypeptides of the present invention also include sequence variants. Variants include substantially homologous polypeptides encoded by the same locus in an organism, ie allelic variants, as well as other variants. Variants are also derived from other loci in an organism but have substantial homology to the polypeptide encoded by the HMGB nucleic acid molecule, its complement and portion, or the nucleotide sequence of the HMGB nucleic acid molecule. Also encompassed are polypeptides having substantial homology to the polypeptides encoded by the containing nucleic acid molecules. Examples of HMGB nucleic acid molecules are known in the art and can be derived from HMGB polypeptides as described herein. Variants also include polypeptides that are substantially homologous or identical to these polypeptides but that are derived from other organisms, ie, orthologs. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by recombinant methods. Preferably, the HMGB polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and It has at least 60%, more preferably at least 70%, 75%, 80%, 85% or 90%, most preferably at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 18.

  In another embodiment, the present invention relates to HMGB polypeptide fragments having HMGB biological activity. “HMGB polypeptide fragment having biological activity of HMGB” or “biologically active HMGB fragment” means a fragment of HMGB polypeptide having the activity of HMGB polypeptide. An example of such an HMGB fragment is the HMGB B box described herein. Biologically active HMGB fragments are compared to appropriate controls using standard molecular biology techniques and when the fragments are administered to cells, for example using the methods described herein. Can be made by assessing the function of the fragment by examining whether it increases the release of pro-inflammatory cytokines from the cell.

  As used herein, “HMGB A box” is also referred to herein as “A box” and is a substantially pure or substantially pure and isolated polypeptide. Which is separated from the components that naturally accompany it and consists of an amino acid sequence that is smaller than the full-length HMGB polypeptide and has the following biological activity: inhibition of inflammation, and / or Or one or more of inhibiting the release of pro-inflammatory cytokines from cells and / or reducing the activity of the inflammatory cytokine cascade. In one embodiment, the HMGB A box polypeptide has one of the biological activities described above. In another embodiment, the HMGB A box polypeptide has two of the biological activities described above. In a third embodiment, the HMGB A box polypeptide has all three of the biological activities described above. Preferably, the HMGB A box has no more than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the biological activity of the full length HMGB polypeptide . In one embodiment, the amino acids of the HMGB A box consist of the sequence of SEQ ID NO: 4, SEQ ID NO: 22 or SEQ ID NO: 57, or the amino acid sequence of the corresponding region of the HMGB protein in mammals.

  The HMGB A box is also a recombinantly produced polypeptide having an amino acid sequence similar to the A box sequence described above. Preferably, the HMGB A box is a mammalian HMGB A box, such as a human HMGB A box. The HMGB A box polypeptide of the present invention preferably consists of the sequence of SEQ ID NO: 4, SEQ ID NO: 22 or SEQ ID NO: 57, or the amino acid sequence of the corresponding region of the HMGB protein in mammals. HMGB A boxes often have about 85 amino acids or less and about 4 amino acids or more. Examples of polypeptides having an A box inside are, but are not limited to, the HMGB proteins and polypeptides described herein. The sequence of the A box in such a polypeptide can be determined using the methods described herein, eg, by comparing the sequence with the A box described herein, and according to the methods described herein or the art. Can be measured and isolated by testing the biological activity of the A box using other methods known in.

  Another example of an HMGB A box polypeptide is the following sequence: PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (human HMGB1; SEQ ID NO: 40); DSSVNFAEF SKKCSERWKT MSAKEKSKFE DMAKSDKARY DREMKNYVPP KGDK (human HMGBPE; DREMKDYGPA KGGK (human HMGB3; SEQ ID NO: 42); PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (HMG1L5 (formerly HMG1L10); SEQ ID NO: 43); SDASVNFSEF SNKCSERWKT MSAKEKGKTY DMAAKE1K (HMG1L4; SEQ ID NO: 45); PDASVKFSEF LKKCSETWKT IFAKEKGKFE DMAKADKAHY EREMKTYIPPKGEK (HAC sequence of BAC clone RP11-395A23; SEQ ID NO: 46); PDASINFSEF SQKCPETWKT TIAKEKGKFE DMAKADKAHY EREMKILIPIL 48); PDASVNFSEF SKKCLVRGKT MSAKEKGQFE AMARADKARY EREMKTYIP PKGET (L OC122441; SEQ ID NO: 49); LDASVSFSEF SNKCSERWKT MSVKEKGKFE DMAKADKACY EREMKIYPYL KGRQ (LOC139603; SEQ ID NO: 50); and GKGDPKKPRG KMSSYAFFVQ TCREEHKKKH PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKGET ERMK57

  The HMGB A box polypeptide of the present invention also encompasses sequence variants. Variants include substantially homologous polypeptides encoded by the same locus in an organism, ie allelic variants, as well as other variants. Variants are also derived from other loci in the organism but have substantial homology to polypeptides encoded by HMGB A box nucleic acid molecules, complements and portions thereof, or HMGB A box nucleic acid molecules Also encompassed are polypeptides having substantial homology to polypeptides encoded by nucleic acid molecules comprising the nucleotide sequence of: Examples of HMGB A box nucleic acid molecules are known in the art and can be derived from HMGB A box polypeptides as described herein. Variants also include polypeptides that are substantially homologous or identical to these polypeptides but that are derived from other organisms, ie, orthologs. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by recombinant methods. Preferably, the HMGB A box is determined by the methods described herein or in the art as measured using one or more of the BLAST programs and parameters described herein and the biological activity of the HMGB A box polypeptide. At least 60%, more preferably at least 60% of the HMGB A box polypeptide described herein, e.g., SEQ ID NO: 4, SEQ ID NO: 22, or SEQ ID NO: 57, as measured using other known methods. 70%, 75%, 80%, 85% or 90%, most preferably at least 95% sequence identity.

  The invention also features a biologically active fragment of the A box. “A box fragment with A box biological activity” or “A box biologically active fragment” means a fragment of an HMGB A box having the activity of an HMGB A box as described herein. For example, A box fragments can reduce the release of pro-inflammatory cytokines from vertebrate cells, reduce inflammation, and / or reduce the activity of the inflammatory cytokine cascade. A-box fragments inhibit the release of pro-inflammatory cytokines from cells using standard molecular biology techniques and when the fragments are administered to cells, for example using the methods described herein It can be prepared by evaluating the function of the fragment by examining whether or not. A-box biologically active fragments can be used in the methods described herein in which full-length A-box polypeptides are used, for example, to suppress the release of pro-inflammatory cytokines from cells or to activate the inflammatory cytokine cascade. It can be used in the treatment of patients with a characteristic condition.

  As used herein, “HMGB B box” is also referred to herein as “B box” and is a substantially pure or substantially pure and isolated polypeptide. And is separated from the components that naturally accompany it and consists of an amino acid sequence that is smaller than the full-length HMGB polypeptide and has the following biological activity: increased inflammation, and Or one or more of increased pro-inflammatory cytokine release from cells and / or increased activity of the inflammatory cytokine cascade. In one embodiment, the HMGB B box polypeptide has one of the biological activities described above. In another embodiment, the HMGB B box polypeptide has two of the biological activities described above. In a third embodiment, the HMGB B box polypeptide has all three of the biological activities described above. Preferably, the HMGB B box has at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the biological activity of the full length HMGB polypeptide. In another embodiment, the HMGB B box does not include an HMGB A box.

  In another embodiment, the HMGB B box is about 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25% or 20% of the length of the full length HMGB1 polypeptide. A polypeptide. In another embodiment, the HMGB B box comprises or consists of the sequence of SEQ ID NO: 5, SEQ ID NO: 20 or SEQ ID NO: 58 or the corresponding region of an HMGB protein in a mammal, but from a full-length HMGB polypeptide It is short. The HMGB B box polypeptide is also a recombinantly produced polypeptide having the same amino acid sequence as the HMGB B box polypeptide described above. Preferably, the HMGB B box is a mammalian HMGB B box, such as a human HMGB B box. The HMGB B box often has about 85 amino acids or less and about 4 amino acids or more. Examples of polypeptides having an internal B box are, but are not limited to, the HMGB proteins and polypeptides described herein. The sequence of the B box in such a polypeptide can be determined using the methods described herein, for example, by comparison of the sequence with the B box described herein, and in the methods described herein or in the art. Can be measured and isolated by testing biological activity using methods known in the art.

  Additional examples of HMGB B box polypeptide sequences include the following sequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (human HMGB1; SEQ ID NO: 51); KKDPNAPKRP PSAFFLFCSE KRP; YRPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMG1L5 (previous HMG1L10); SEQ ID NO: 53); FKDPNAPKRP PSAFFLFCSE YHPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPGEKKA AKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 54); FKDSNAPKRP PSAFLLFCSE YCPKIKGEHP GLPISDVAKK LVEMWNNTFA DDKQLCEKKA AKLKEKYKKD TATY (HMG1L4; SEQ ID NO: 55) FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVVKK LAGMWNNTAA ADKQFYEKKA AKLKEKYKKD IAAY (HMG sequence of BAC clone RP11-359A23; SEQ ID NO: 56);

  The HMGB B box polypeptide of the present invention also encompasses sequence variants. Variants include substantially homologous polypeptides encoded by the same locus in an organism, ie allelic variants, as well as other variants. Variants are also derived from other loci in the organism but have substantial homology to polypeptides encoded by HMGB box nucleic acid molecules, complements and portions thereof, or of HMGB B box nucleic acid molecules. Also encompassed are polypeptides having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence. Examples of HMGB B box nucleic acid molecules are known in the art and can be derived from the HMGB B box polypeptides described herein. Variants also include polypeptides that are substantially homologous or identical to these polypeptides but that are derived from other organisms, ie, orthologs. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides produced by recombinant methods. Preferably, the non-natural HMGB B box polypeptide, as measured using the BLAST program and parameters described herein, is the sequence of the HMGB B box described herein, eg, SEQ ID NO: 5, SEQ ID NO: 20, or It has at least 60%, more preferably at least 70%, 75%, 80%, 85% or 90%, most preferably at least 95% sequence identity to the sequence of SEQ ID NO: 58. Preferably, the HMGB B box consists of the sequence of SEQ ID NO: 5, SEQ ID NO: 20 or SEQ ID NO: 58, or the amino acid sequence of the corresponding region of the HMGB protein in a mammal, and the methods described herein or in the art It has one or more of the biological activities of the HMGB B box as measured using other known methods.

  In another embodiment, the present invention relates to a polypeptide comprising an HMGB B box biologically active fragment having a B box biological activity, or a non-natural HMGB B box fragment. In another aspect, the invention relates to a vertebrate HMGB B box or a fragment thereof having biological activity of a B box, or a polypeptide comprising a non-natural HMGB B box but not a full-length HMGB polypeptide. . “B box fragment having B box biological activity” or “B box biologically active fragment” means an HMGB B box fragment having HMGB B box activity. For example, a B box fragment can induce the release of proinflammatory cytokines from vertebrate cells, increase inflammation, or induce an inflammatory cytokine cascade. An example of such a B box fragment is a fragment (SEQ ID NO: 16 or SEQ ID NO: 23) comprising the first 20 amino acids of the HMGB1B box described herein. B box fragments are suitable when the fragment is administered to a cell using standard molecular biology techniques and using, for example, the methods described herein or other methods known in the art. Can be made by assessing the function of the fragment by examining whether it increases the release of pro-inflammatory cytokines from the cell when compared to a normal control.

  HMGB polypeptides, HMGB A box and HMGB B box, either natural or non-natural, include polypeptides having sequence identity with the HMGB polypeptides, HMGB A boxes and HMGB B boxes described herein. To do. As used herein, two polypeptides (or regions of polypeptides) have an amino acid sequence of at least about 60%, 70%, 75%, 80%, 85%, 90% or 95% or more, homologous or If they are identical, they are substantially homologous or identical. The percent identity between two amino acid sequences (two or nucleic acid sequences) can be determined by aligning the sequences for optimal comparison purposes (eg, gaps can be introduced into the sequence of the first sequence). The amino acids or nucleotides at the 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 (ie,% identity = number of identical positions / Total number of positions x 100). In certain embodiments, the length of an HMGB polypeptide, HMGB A box polypeptide or HMGB B box polypeptide aligned for comparison purposes is shown in a comparative sequence, eg, FIGS. 12A-12E, 14A-14P. At least 30%, preferably at least 40%, more preferably at least 60%, more preferably at least 70%, 80%, 90% or 100% of the length of the sequence and SEQ ID NOs: 18, 20, and 22. The actual comparison of the two sequences can be done in a well-known manner using, for example, a mathematical algorithm. A preferred 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) 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 (eg, BLASTN) can be used. You can refer to the Internet site of the National Center for Biotechnology Information (NCBI). In one embodiment, the database to be searched is a non-redundant (NR) database and the parameters for sequence comparison are unfiltered; expectation value is 10; Word Size is 3; Matrix is BLOSUM62; and Gap Cost is Existence Can be set to 11 and Extension has 1.

  Another preferred non-limiting example of a mathematical algorithm that can be used for sequence comparison 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 weightless 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 are described in Torellis and Robotti (Comput. Appl. Biosci., 10: 3-5, 1994) ADVANCE and ADAM; and Pearson and Lipman (Proc. Natl Acad. Sci USA, 85: 2444-2448, 1988).

  In another embodiment, the percent identity between two amino acid sequences is calculated 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. , Using the GAP program in the GCG software package (Accelrys, San Diego, California). In yet another embodiment, the percent identity between two nucleic acid sequences may be performed using the GAP program in the GCG software package (Accelrys, San Diego, California) using gap weight 50 and length weight 3. it can.

  A “cytokine” as used herein is a soluble protein or peptide that is naturally produced by mammalian cells and functions in vivo as a humoral regulator at micro to picomolar concentrations. Cytokines modulate the functional activity of individual cells and tissues either under normal or pathological conditions. Pro-inflammatory cytokines are any of the physiological reactions associated with inflammation described below: vasodilation, hyperemia, increased vascular permeability associated with edema, granulocyte and mononuclear phagocyte accumulation, or fibrin adhesion It is a cytokine that can induce. In some cases, pro-inflammatory cytokines can also induce apoptosis, such as in the case of chronic heart disease where TNF is known to promote cardiomyocyte apoptosis (Pulkki, Ann. Med. 29: 339-343, 1997; and Tsutsui et al., Immunol. Rev. 174: 192-209, 2000).

  Non-limiting examples of proinflammatory cytokines include tumor necrosis factor (TNF), interleukin (IL) -1α, IL-1β, IL-6, IL-8, IL-18, interferon γ, HMG-1, platelets Activator (PAF) and macrophage migration inhibitory factor (MIF).

  Pro-inflammatory cytokines must be distinguished from anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 which are not mediators of inflammation.

  In many cases, pro-inflammatory cytokines are produced in an inflammatory cytokine cascade as defined herein as in vivo release of at least one pro-inflammatory cytokine in a mammal where the release of the cytokine affects the physiological state of the mammal. That is, the inflammatory cytokine cascade is suppressed in aspects of the invention where the release of pro-inflammatory cytokines induces deleterious physiological conditions.

  As used herein, an “agent that inhibits the biological activity of TNF” is an agent that reduces one or more of the biological activities of TNF. Examples of TNF biological activity include, but are not limited to, vasodilation, hyperemia, increased vascular permeability associated with edema, granulocyte and mononuclear phagocyte accumulation, or fibrin adhesion. Agents that suppress TNF biological activity include agents that suppress (reduce) the interaction between TNF and the TNF receptor. Examples of such agents are antibodies that bind TNF or antigen-binding fragments thereof, antibodies that bind to TNF receptors or antigen-binding fragments thereof, and bind to TNF or TNF receptors to prevent TNF / TNF receptor interactions Including molecules that Examples of such agents include, but are not limited to, peptides, proteins, synthetic molecules such as synthetic organic molecules, natural molecules such as natural organic molecules, nucleic acid molecules and components thereof. Preferred examples of agents that suppress the biological activity of TNF include infliximab (Remicade; Centocor, Inc., Malvern, Pennsylvania), etanercept (Immunex; Seattle, Washington), adalimumab (D2E7; Abbot Laboratories, Abbot Park Illinois), Includes CDP870 (Pharmacia Corporation; Bridgewater, New Jersey), CDP571 (Celltech Group plc, United Kingdom), Renercept (Roche, Switzerland) and thalidomide.

  The inflammatory cytokine cascade contributes to deleterious properties including inflammation and apoptosis in many diseases. Included in these are disorders characterized by both local and systemic reactions, such as, but not limited to, disorders described herein (eg, conditions described in the background art herein). Specific disorders characterized by an inflammatory cytokine cascade include, for example, sepsis, allograft rejection, rheumatoid arthritis, asthma, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial illness Includes blood, organ ischemia, reperfusion ischemia, Behcet's disease, graft-versus-host disease, Crohn's disease, ulcerative colitis, multiple sclerosis and cachexia.

A-box polypeptides and biologically active fragments thereof As described herein, in one aspect, the present invention can inhibit the release of pro-inflammatory cytokines from cells treated with HMG, or the inflammatory cytokine cascade It relates to a polypeptide composition comprising a vertebrate HMGB A box or biologically active fragment thereof, which can be used to treat conditions characterized by activation. In certain embodiments, the present invention relates to an agent that inhibits the biological activity of TNF, such as infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide in combination with one or more HMGB A boxes or biological activity thereof. It relates to a composition comprising a fragment or variant. Such compositions can be used to inhibit the release of pro-inflammatory cytokines from vertebrate cells administered HMG and / or can be used to treat conditions characterized by activation of the inflammatory cytokine cascade .

  When referring to the effect of any of the compositions or methods of the invention on the release of proinflammatory cytokines, the term “suppressing” or “reducing” means at least a small but measurable reduction in proinflammatory cytokine release. Is included. In preferred embodiments, pro-inflammatory cytokine release is suppressed by at least 20% over untreated controls; in more preferred embodiments, inhibition is at least 50%; in even more preferred embodiments, inhibition is at least 70%. And in a most preferred embodiment, the inhibition is at least 80%. Inhibition can be assessed using the methods described herein or other methods known in the art. Such a reduction in pro-inflammatory cytokine release can reduce the deleterious effects of the inflammatory cytokine cascade in an in vivo manner.

  Because HMGB A boxes (eg, vertebrate HMGB A boxes) have a high level of sequence conservation (see, eg, Figure 13 for comparison of amino acid sequences of rat, mouse and human HMGB polypeptides), the HMGB A box (Eg, vertebrate HMGB A box) is thought to be able to inhibit the release of proinflammatory cytokines from vertebrate cells treated with HMGB. Accordingly, HMGB A boxes (eg, vertebrate HMGB A boxes) are included within the scope of the present invention. Preferably, the HMGB A box is a vertebrate HMGB A box (eg, a mammalian HMGB A box, eg, a human HMGB1 A box set forth herein as SEQ ID NO: 4, SEQ ID NO: 22, or SEQ ID NO: 57). Furthermore, the present invention also encompasses fragments of HMGB1 A box having the biological activity of HMGB A box as described herein.

  As one skilled in the art knows, a non-natural HMGB A box (or biologically active fragment thereof) can be generated without undue experimentation, from vertebrate cells treated with vertebrate HMGB. Inhibits the release of pro-inflammatory cytokines. Such a non-natural functional A box (variant) aligns the amino acid sequence of the HMGB A box from various sources, and the A box causes one or more substitutions at one of the sequences at different amino acid positions Can be produced. Substitutions are preferably made using the same amino acid residues that are present in the A box to be compared. Alternatively, conservative substitutions are made from any of the residues.

  Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains. For example, one group of amino acids is an amino acid with neutral and hydrophobic side chains (a, v, l, i, p, w, f and m); another group has neutral and polar side chains (G, s, t, y, c, n and q); another group is an amino acid having a basic side chain (k, r and h); another group has an acidic side chain (D and e); another group is an amino acid having an aliphatic side chain (g, a, v, l and i); another group is an amino acid having an aliphatic hydroxyl side chain ( s and t); another group is amino acids with amine-containing side chains (n, q, k, r and h); another group is amino acids with aromatic side chains (f, y and w) And another group are amino acids with sulfur-containing side chains (c and m). Preferred conservative amino acid substitution groups are rk, ed, yf, l-m, v-i and q-h.

Although conservative amino acid substitutions are expected to retain the biological activity of the HMGB A box polypeptide, the following is a representation of the human HMGB1 A box (SEQ ID NO: 4) of SEQ ID NO: 3 of the human HMGB2 A box. An example of how a non-natural A-box polypeptide (variant) can be made by comparison with residues 32-85 (SEQ ID NO: 17).
HMGB1 pdasvnfsef skkcserwkt msakekgkfe dmakadkary eremktyipp kget (SEQ ID NO: 4);
HMGB2 pdssvnfaef skkcserwkt msakekskfe dmaksdkary dremknyvpp kgdk (SEQ ID NO: 17).

  The non-natural HMGB A box can be prepared, for example, by replacing the alanine (a) residue at the 3rd position of the HMGB1 A box with the serine (s) residue present at the 3rd position of the HMGB2 A box. As the skilled artisan knows, the substitution gives a functional non-natural A box because the s residue can function at that position of the HMGB2 A box. Alternatively, position 3 of the HMGB1 A box can be substituted with any amino acid conserved with respect to alanine or serine, such as glycine (g), threonine (t), valine (v) or leucine (l). As those skilled in the art will appreciate, such substitutions are functional because the A box is not invariant at position 3 and conservative substitutions provide sufficient structural alternatives to naturally occurring amino acids at that position. Expected to bring a target A box.

  According to the method described above, a large number of non-natural HMGB A boxes can be made without particularly unscheduled experiments, which are expected to be functional and any particular non-natural NGGB The functionality of the HMGB A box can be predicted with sufficient accuracy. In any case, the functionality of any non-natural HMGB A box is simply added to the cell along with the HMGB polypeptide, and the release of proinflammatory cytokines by the cell using, for example, the methods described herein. By examining whether the A box suppresses, it can be investigated without any unplanned experiments.

  The cell in which the A box or biologically active fragment of the A box inhibits the release of HMG-induced proinflammatory cytokines can be any cell that can be induced to produce proinflammatory cytokines. In preferred embodiments, the cells are mammalian cells, such as immune cells (eg, macrophages, monocytes or neutrophils).

  Polypeptides comprising A-boxes or biologically active fragments of A-boxes that are capable of suppressing the production of any single pro-inflammatory cytokine now known or later discovered are within the scope of the present invention. Is included. Preferably, the antibody can suppress the production of TNF, IL-1β and / or IL-6. Most preferably, the antibody can inhibit the production of any pro-inflammatory cytokine produced by vertebrate cells.

B box polypeptides and biologically active fragments thereof As described herein, in one aspect, the present invention relates to polypeptide compositions comprising vertebrate HMGB B boxes or biologically active fragments thereof, which include HMGB Release of proinflammatory cytokines from vertebrate cells administered with

  When referring to the effect of any of the compositions or methods of the invention on the release of proinflammatory cytokines, use of the term “increase” includes at least a slight but measurable increase in proinflammatory cytokine release. . In preferred embodiments, the release of proinflammatory cytokines is increased by at least 1.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold compared to an untreated control. Such increased pro-inflammatory cytokine release can increase the action of the inflammatory cytokine cascade in an in vivo manner. Such polypeptides can also be used for inducing weight loss and / or treating obesity.

  Because all HMGB B boxes exhibit a high level of sequence conservation (see, eg, Figure 13 for comparison of amino acid sequences of rat, mouse and human HMGB polypeptides), functional non-natural HMGB B boxes are functional. Make one or more conservative amino acid substitutions as described above for creating a non-natural A box, or compare naturally occurring vertebrate B boxes from different sources and replace related amino acids By doing so, it can be produced without conducting an unscheduled experiment. In a particularly preferred embodiment, the B box comprises the sequence of human HMGB1B box (3 different lengths) SEQ ID NO: 5, SEQ ID NO: 20 or SEQ ID NO: 58, or in the polypeptide shown in FIGS. A fragment of the HMGB B box that contains the derived B box sequence or has the biological activity of the B box. For example, the 20 amino acid sequence contained in SEQ ID NO: 20 contributes to the function of the B box. This 20 amino acid B box fragment has the following amino acid sequence: fkdpnapkrl psafflfcse (SEQ ID NO: 23). Another example of a biologically active fragment of the HMGB B box consists of amino acids 1-20 of SEQ ID NO: 5 (napkrppsaf flfcseyrpk; SEQ ID NO: 16).

Antibodies against HMGB and HMGB B box polypeptides The present invention also relates to purified preparations of antibodies that bind to HMGB polypeptides or biologically active fragments thereof (anti-HMGB antibodies). The anti-HMGB antibody can be a neutralizing antibody (ie inhibits the biological activity of an HMG polypeptide or biologically active fragment thereof, eg, the release of proinflammatory cytokines from vertebrate cells induced by HMG be able to). The present invention also provides antibodies (anti-HMGB B) that specifically bind to a vertebrate high mobility group protein (HMG) B box or biologically active fragment thereof but do not selectively bind to non-B box epitopes of HMGB. Box antibody). In these embodiments, the antibody can also be a neutralizing antibody (ie, they are from a biological activity of a B box polypeptide or biologically active fragment thereof, eg, from vertebrate cells induced by HMGB. Can inhibit the release of pro-inflammatory cytokines). Such antibodies can be combined with one or more agents that inhibit the biological activity of TNF, such as infliximab, etanercept, adalimumab, CDP870, CDP571, renercept or thalidomide.

The term “antibody” or “purified antibody” as used herein includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, antigen binding sites that selectively bind antigen. Refers to a molecule. A molecule that selectively binds to a polypeptide of the invention binds to the polypeptide or a fragment thereof, but does not substantially bind to other molecules in the sample, eg, a biological sample that naturally contains the polypeptide. Is a molecule. Preferably the antibody is at least 60% by weight free of proteins and natural organic molecules with which it is naturally associated. More preferably the antibody preparation is at least 75% or 90%, most preferably 99% by weight antibody. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ′) ₂ fragments that can be generated by treating the antibody with an enzyme such as pepsin.

  The present invention provides polyclonal and monoclonal antibodies that selectively bind to the HMGB B box polypeptides of the present invention. The term “monoclonal antibody” or “or composition” as used herein refers to an antibody molecule that contains only a single species of antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. Refers to a group of people. That is, a monoclonal antibody composition typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.

  Polyclonal antibodies can be prepared, for example, by immunizing a suitable subject with a desired immunogen, eg, an HMGB B box polypeptide of the present invention or a fragment thereof, as described herein. The antibody titer of the immunized subject can be monitored over time by standard techniques such as an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the IgG fraction can be obtained by isolating antibody molecules directed against the polypeptide from a mammal (eg, blood) and further purifying by well-known techniques such as protein A chromatography.

  At an appropriate time after immunization, for example, when the antibody titer is at its highest value, antibody-producing cells are obtained from the subject and used to calculate Kohler and Milstein (Nature 256: 495-497, 1975), the hybridoma method described first, the human B cell hybridoma method (Kozbor et al., Immunol. Today 4:72, 1983), the EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or monoclonal antibodies can be prepared by standard techniques such as the trioma method. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, Coligan et al., (Ed.) John Wiley & Sons, Inc., New York, NY, 1994). Briefly, an immortal cell line (typically myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above and the resulting hybridoma The cell culture supernatant is screened to identify hybridomas that produce monoclonal antibodies that bind to specific polypeptides (eg, polypeptides of the invention).

  Any of a number of known protocols for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating monoclonal antibodies to the polypeptides of the invention (eg Current Protocols in Immunology, supra; Galfre et al. (Nature, 266: 55052, 1977); RH Kenneth, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); Lerner (Yale J. Biol. Med. 54: 387-402, 1981). Furthermore, there are many variations of such methods that are equally useful as those skilled in the art know.

  In one alternative method of preparing a monoclonal antibody-secreting hybridoma, a monoclonal antibody against the HMGB B box polypeptide of the present invention is obtained by screening a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) using the polypeptide. By isolating an immunoglobulin library member that binds to the polypeptide, it can be discovered and isolated. Kits for generating and screening phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP ™ Phage Display Kit, catalog number 240612). Furthermore, examples of methods and reagents that are particularly suitable for preparing and screening antibody display libraries include, for example, US Pat. No. 5,223,409; International Publication No. WO92 / 18619; International Publication No. WO91 / 17271; International Publication No. WO92 / International Publication No. WO92 / 15679; International Publication No. WO93 / 01288; International Publication No. WO92 / 01047; International Publication No. WO92 / 09690; International Publication No. WO90 / 02809; Fuchs et al., Bio / Technology 9 : 1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3: 81-85, 1992; Huse et al. (Science 246: 1275-1281, 1989); Griffiths et al. (EMBO J. 12: 725-734, 1993) Are listed.

  Furthermore, recombinant antibodies such as chimeric or humanized monoclonal antibodies comprising both human and non-human portions that can be generated using standard recombinant DNA techniques are also encompassed within the scope of the present invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

  In general, antibodies of the invention (eg, monoclonal antibodies) can be used to isolate HMGB B box polypeptides of the invention by standard techniques such as affinity chromatography or immunoprecipitation. Polypeptide-specific antibodies facilitate the purification of native polypeptides from cells and recombinantly produced polypeptides expressed in host cells. Furthermore, the amount and pattern of polypeptide expression can be assessed by detecting the polypeptide (eg, in a cell lysate, cell supernatant or tissue sample) using an antibody specific for the HMGB B box polypeptide of the present invention. can do.

  Because vertebrate HMGB polypeptides and HMGB B boxes exhibit a high level of sequence conservation, vertebrate HMGB polypeptides or HMGB B boxes are generally thought to be capable of inducing pro-inflammatory cytokine release from vertebrate cells. It has been. Accordingly, antibodies to vertebrate HMGB polypeptides or HMGB B boxes are included within the scope of the present invention. In one embodiment, the antibody is a neutralizing antibody.

  Preferably, the HMGB polypeptide is a mammalian HMG as described herein, more preferably a mammalian HMGB1 polypeptide, most preferably a human HMGB1 polypeptide set forth herein as SEQ ID NO: 1. The antibody can also be directed against an HMGB polypeptide fragment having the biological activity of the HMGB polypeptide.

  Preferably, the HMGB B box is a mammalian HMGB B box, more preferably a mammalian HMGB1B box, and most preferably a human HMGB1B box shown herein as SEQ ID NO: 5, SEQ ID NO: 20 or SEQ ID NO: 58. The antibody can also be directed against an HMGB B box fragment having B box biological activity.

  Antibodies raised against an HMGB immunogen or HMGB B box immunogen can include HMGB polypeptides or fragments thereof, HMGB B boxes or fragments thereof, or animals containing HMGB polypeptides, HMGB B boxes or fragments thereof, Preferably, it can be obtained by administering to a non-human using a routine protocol. Mice or other animals, such as rats or chickens, are immunized by using as an antigen a polypeptide such as an antigenic or immunologically equivalent derivative. The immunogen can be associated with an immunogenic carrier protein, such as bovine serum albumin (BSA) or mussel hemocyanin (KLH), for example, by conjugation. Alternatively, multi-antigen peptides comprising multiple copies of HMGB or HMGB B boxes or fragments are sufficiently antigenic to improve immunogenicity and do not require a carrier. Bispecific antibodies having two antigen-binding domains, each directed against a different HMGB or HMGB B box epitope, may also be produced by routine methods.

  For the preparation of monoclonal antibodies, any method known in the art that provides antibodies produced by continuous cell line cultures can be used. See, for example, Kohler and Milstein, supra; and Cole et al., Supra.

  Techniques for the production of single chain antibodies (US Pat. No. 4,946,778) can be employed to produce single chain antibodies against HMGB, B box or fragments thereof. Furthermore, humanized antibodies may be expressed using transgenic mice or other organisms, such as other mammals.

  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 a human, the antibody is preferably “humanized”, ie, the complementarity determining region of the antibody is transferred to the human antibody (eg, Jones et al. (Nature 321: 522-525, 1986); and Tempest et al. (Described in Biotechnology 9: 226-273, 1991).

  Antibody genes that have binding activity against polypeptides obtained from a repertoire of PCR amplified v genes from lymphocytes, obtained from humans screened for anti-B-box antibodies or from naive libraries Phage display methods can also be used to select (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).

  Once antibodies that specifically bind to HMGB epitopes or HMGB B box epitopes are obtained, they can then be screened for the ability to suppress the release of proinflammatory cytokines without undue experimentation. . Anti-antigen capable of inhibiting the production of any single pro-inflammatory cytokine and / or inhibiting the release of pro-inflammatory cytokines from cells and / or conditions characterized by activation of the inflammatory cytokine cascade HMGB B box antibodies are within the scope of the present invention. Preferably, the antibody can suppress the production of TNF, IL-1β and / or IL-6. Most preferably, the antibody can inhibit the production of any pro-inflammatory cytokine produced by vertebrate cells.

  For methods of inhibiting the release of pro-inflammatory cytokines from cells using antibodies to HMGB B boxes or biologically active fragments thereof, or for treating conditions characterized by activation of the inflammatory cytokine cascade, The cell can be any cell that can be induced to produce pro-inflammatory cytokines. In preferred embodiments, the cells are immune cells such as macrophages, monocytes or neutrophils.

A composition comprising one or more of an HMGB A box polypeptide, an antibody against HMGB, an antibody against HMGB B box, and an inhibitor of TNF biological activity. In certain embodiments, the present invention is in a pharmaceutically acceptable carrier. Relates to a composition comprising any of the above polypeptides (eg HMGB A box polypeptide or biologically active fragment according to the invention). Suitable pharmaceutically acceptable carriers include those described herein. In these embodiments, the composition can inhibit conditions characterized by activation of the inflammatory cytokine cascade. The condition can be one in which the inflammatory cytokine cascade induces a systemic response such as endotoxin shock. Alternatively, the condition can be mediated by a localized inflammatory cytokine cascade as in rheumatoid arthritis. Non-limiting examples of conditions that can be usefully treated by the present invention include those listed in the background section of this specification. In one embodiment, the condition being treated is appendicitis, peptic ulcer, gastric ulcer and duodenal ulcer, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, Anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic miscarriage, disseminated bacteremia, burn, Alzheimer's disease, celeriac disease, congestive Includes heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection, graft-versus-host disease. In another embodiment, the condition includes endotoxin shock or allograft rejection. Where the condition is allograft rejection, the composition may conveniently contain an immunosuppressant used to suppress allograft rejection, such as cyclosporine.

  In another aspect, the invention relates to a composition comprising an antibody preparation as described above (eg, an anti-HMGB B box antibody or a biologically active fragment thereof as described herein) in a pharmaceutically acceptable carrier. In these embodiments, the composition can inhibit conditions characterized by activation of the inflammatory cytokine cascade. Conditions that can be treated with these compositions are as listed above.

  In another embodiment, the present invention provides an HMGB A box polypeptide as described above, and / or an antibody or antigen-binding fragment thereof that binds to HMGB, and / or an antibody or antigen-binding fragment thereof that binds to an HMGB B box, and TNF The present invention relates to compositions comprising any of the agents that inhibit biological activity (collectively referred to as “combination therapy compositions”). Preferred examples of agents that inhibit TNF biological activity are infliximab, etanercept, adalimumab, CDP870, CDP571, renercept and thalidomide. Such combination therapy compositions can further comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include those described herein. In these embodiments, the combination therapy composition is capable of inhibiting conditions characterized by activation of the inflammatory cytokine cascade and / or inhibiting the release of proinflammatory cytokines from cells. The condition can be one in which the inflammatory cytokine cascade induces a systemic response such as endotoxin shock. Alternatively, the condition can be mediated by a localized inflammatory cytokine cascade as in rheumatoid arthritis. Non-limiting examples of conditions that can be usefully treated by the present invention include those listed in the background section of this specification. In one embodiment, the condition to be treated is sepsis, allograft rejection, rheumatoid arthritis, asthma, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, Includes organ ischemia, reperfusion ischemia, Behcet's disease, graft-versus-host disease, Crohn's disease, ulcerative colitis, multiple sclerosis and cachexia. Preferably, the combination therapy composition is in an amount sufficient to inhibit the release of proinflammatory cytokines from the cells and / or to treat a condition characterized by activation of the inflammatory cytokine cascade To be administered. In one embodiment, pro-inflammatory cytokine release is at least 10%, 20%, 25%, 50% when assessed using the methods described herein or other methods known in the art. 75%, 80%, 90% or 95% suppressed.

  Polypeptide (eg HMGB A box polypeptide or biologically active fragment thereof), antibody (eg anti-HMGB B box antibody or biologically active fragment thereof) or combination therapy composition (eg HMGB A box polypeptide or biological thereof) Active fragment and agent that suppresses TNF biological activity, and / or antibody that binds to HMGB or antigen-binding fragment thereof and agent that suppresses TNF biological activity, and / or antibody that binds to HMGB B box Or an antigen-binding fragment thereof and an agent that suppresses TNF biological activity) is selected based on the expected route of administration of the composition in therapeutic use. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection is preferred for the treatment of systemic disorders such as endotoxin shock, and oral administration is preferred for the treatment of gastrointestinal disorders such as gastric ulcers. The route of administration and the dose of the composition to be administered can be determined by one skilled in the art without undue experimentation by standard dose-response studies. The relevant circumstances to consider when making such a determination include the condition 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. That is, depending on the condition, the antibody composition can be administered to a patient orally, parenterally, nasally, vaginally, rectally, lingually, sublingually, buccally, buccally and transdermally.

  Thus, a composition designed for oral, lingual, sublingual, buccal, buccal administration is unscheduled by means well known in the art, for example using an inert diluent or edible carrier. It can be manufactured without performing the experiment. The composition may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical composition of the invention is formulated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Good.

  Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agents, lubricants, sweetening agents, and flavoring agents. Some examples of binders are microcrystalline cellulose, gum tragacanth and gelatin. Examples of excipients include starch and lactose. Some examples of disintegrants include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate and potassium stearate. An example of a glidant is colloidal silicon oxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavor and the like. The materials used in preparing these various compositions must be pharmaceutically pure and non-toxic in the amounts used.

  The compositions of the present invention can be readily administered parenterally, for example, intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compositions of the present invention into a solution or suspension. Such solutions or suspensions may also contain sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol and / or other synthetic solvents. Parenteral formulations may also contain antibacterial agents such as benzyl alcohol, and / or methyl paraben, antioxidants such as ascorbic acid and / or sodium bisulfite, and chelating agents such as EDTA. Buffers such as acetate, citrate and / or phosphate and osmotic regulators such as sodium chloride and / or dextrose may also be added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Rectal administration includes administering the pharmaceutical composition into the rectum or large intestine. This can be done using suppositories or enemas. Suppository formulations can be readily prepared by methods known in the art. For example, suppository preparations are prepared by heating glycerin to about 120 ° C., dissolving the polypeptide composition, antibody composition and / or combination therapy composition in glycerin, mixing the heated glycerin, and then adding purified water. It can be prepared by pouring the hot mixture into a suppository mold.

  Transdermal administration includes percutaneous absorption of the composition via the skin. Transdermal formulations include patches, ointments, creams, gels, salves and the like.

  The invention includes nasally administering to a mammal (eg, a human) a therapeutically effective amount of the composition. As used herein, nasal or intranasal administration includes administering the composition to the mucous membrane of the patient's nasal passage or nasal cavity. As used herein, pharmaceutical compositions for intranasal administration of the compositions were prepared in a well-known manner for administration as, for example, nasal sprays, nasal drops, suspensions, gels, ointments, creams or powders. Contains a therapeutically effective amount of a polypeptide, antibody and / or combination therapy. Administration of the composition may be performed using a nasal tampon or nasal sponge.

  The pharmaceutical compositions (eg, polypeptide compositions, antibody compositions and / or combination therapy compositions) described herein also include early sepsis mediator antagonists. As used herein, any early sepsis mediator is a pro-inflammatory cytokine that is released from cells immediately (ie within 30-60 minutes) after induction of the inflammatory cytokine cascade (eg, exposure to LPS). Non-limiting 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 (eg, tumor necrosis factor receptor type 1) and enzymes required for the production of these cytokines, eg, interleukin-1β converting enzyme . Antagonists of early sepsis mediators now known or later discovered are useful for these embodiments by further inhibiting the inflammatory cytokine cascade.

  Non-limiting examples of early sepsis mediator antagonists include antisense compounds that bind to and prevent expression of early sepsis mediator mRNA (eg, Ojwang et al. (Biochemistry 36: 6033-6045, 1997); Pampfer et al. (Biol. Reprod. 52: 1316-1326, 1995); US Pat. No. 6,228,642; Yahata et al. (Antisense Nucleic Acid Drug Dev. 6: 55-61, 1996); and Taylor et al., (Antisense Nucleic Acid Drug Dev. 8: 199- 205, 1998)), a ribozyme that specifically cleaves the mRNA of the early sepsis mediator (eg Leavitt et al., (Antisense Nucleic Acid Drug Dev. 10: 409-414, 2000) Hendrix et al., (Biochem. J. 314 (Pt .2): 655-661, 1996)), and antibodies that bind to the early sepsis mediator and suppress its action (eg Kam and Targan (Expert Opin. Pharmacother. 1: 615-622, 2000); Nagahira et al., (J. Immunol. Methods 222, 83-92, 1999); Lavine et al. (J. Cereb. Blood Flow Metab. 18: 52-58, 1998); Fine Holmes et al (Hybridoma 19: 363-367, 2000) is a reference). Any of the antagonists of early sepsis mediators now known or later discovered are within the scope of the present invention. One skilled in the art can determine the amount of early sepsis mediator for use in these compositions to inhibit any particular inflammatory cytokine cascade without undue experimentation by routine dose-response studies .

Other agents that can be administered with the compositions described herein include, for example, Vitaxin ™ and other antibody targeting α _v β ₃ integrins (eg, US Pat. No. 5,753,230, International Publication Nos. WO 00/78815 and WO 02). / 70007; incorporated herein by reference in its entirety) and anti-IL-9 antibodies (see, eg, WO 97/08321; incorporated herein by reference in its entirety) . Other agents that can be administered with the polypeptide compositions described herein are, for example, B7 antagonists (eg, CTLA4Ig, anti-CD80 antibody, anti-CD86 antibody), methotrexate, and / or CD40 antagonists (eg, anti-CD40 ligand). (CD40L)) (see, eg, Saito et al., J. Immunol. 160 (9): 4225-31 (1998)).

  In a further aspect, the present invention also relates to a method of inhibiting the release of proinflammatory cytokines from mammalian cells. The method comprises any of the HMGB A box compositions described above, and / or any of the compositions of HMGB B box or HMGB B box biologically active fragment antibody, and / or any combination therapy composition. Treating the cells with. This method is believed to be useful for inhibiting the release of cytokines from any mammalian cell that produces proinflammatory cytokines. However, in a preferred embodiment, the cell is a macrophage because macrophage production of the pro-inflammatory cytokine is associated with several important diseases.

  This method is useful for the suppression of any pro-inflammatory cytokine produced by mammalian cells. In preferred embodiments, the proinflammatory cytokines are TNF, IL-1α, IL-1β, MIF and / or IL-6 because these proinflammatory cytokines are particularly important mediators of the disease. is there.

  The methods of these embodiments are useful for in vitro applications, such as in studies to measure the biological characteristics of pro-inflammatory cytokine production in cells. A preferred embodiment, however, is an in vivo therapeutic application where cells are present in patients suffering from or at risk for conditions characterized by activation of the inflammatory cytokine cascade.

  In certain embodiments, the present invention relates to a method of treating a condition in a patient characterized by activation of an inflammatory cytokine cascade. Methods include any of the HMGB A box compositions (including non-natural A box polypeptides and A box biologically active fragments), antibodies of any HMGB B box or B box biologically active fragments described above. This includes administering to the patient a composition (including a non-natural B-box polypeptide or biologically active fragment thereof) and / or any combination therapy composition. This method is expected to be useful for any condition mediated by an inflammatory cytokine cascade, including any of those previously listed. As with the in vivo methods described above, preferred conditions are appendicitis, peptic ulcer, gastric and duodenal ulcer, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, hepatitis, Crohn's disease, asthma, Allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, sepsis, endotoxin shock, cachexia, septic abortion, disseminated bacteremia, burn, Alzheimer's disease, cerebral infarction, brain Includes embolism, spinal cord injury, paralysis, allograft rejection, or graft versus host disease. In the most preferred embodiment, the condition is endotoxin shock or allograft rejection. Where the condition is allograft rejection, the composition may also conveniently contain an immunosuppressive agent used to suppress allograft rejection, such as cyclosporine.

The method of this like also antagonists of early sepsis cis mediators, anti-alpha _v beta ₃ antibody, anti-IL-9 antibodies, B7 antagonists (eg CTLA4Ig, anti-CD80 antibody, anti-CD86 antibody), methotrexate, and / or CD40 antagonist Administration of an agent (eg, an anti-CD40 ligand (CD40L)) can be usefully included. The properties of these drugs are as described above.

  The B-box polypeptides and biologically active fragments thereof described herein can be used for induction of inflammatory cytokines in suitable isolated cells in vitro or ex vivo or as an in vivo treatment. In any of these treatments, the polypeptide or fragment is suitably operably linked to a B box or B box fragment encoded to be synthesized in the cell or patient in which the B box or B box fragment is treated. The control sequence can be administered by providing it to a DNA or RNA vector encoding a B box or B box fragment. In vivo applications include the use of B box polypeptides or B box fragment polypeptides or vectors as a treatment for weight loss. See WO 00/47104, which reveals that treatment with HMGB1 induces weight loss (incorporated herein in its entirety by reference).

  In certain embodiments, the present invention relates to methods for stimulating the release of proinflammatory cytokines from cells. The method may be a B box polypeptide or a biologically active B box fragment polypeptide, eg, the sequence of SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 58, SEQ ID NO: 16 or SEQ ID NO: 23 as described herein. Treating the cells with any of the polypeptides comprising or consisting of these (including non-natural B-box polypeptides and fragments). This method is useful for in vitro applications, such as examining the effects of pro-inflammatory cytokine production on the biological characteristics of the production cells. Since the HMGB B box has the activity of HMGB protein, the B box is also expected to induce weight loss. Accordingly, in a further aspect, the invention is a method for effecting weight loss or treating obesity in a patient. The method includes administering to the patient an effective amount of any of the B box polypeptides or B box fragment polypeptides described herein (including non-natural B box polypeptides and fragments). In another embodiment, the B box polypeptide or B box fragment polypeptide is in a pharmaceutically acceptable carrier.

Screening for Modulators of Proinflammatory Cytokine Release from Cells The present invention also relates to methods for examining whether a compound (test compound) inhibits inflammation and / or inflammatory response. The method combines (a) a cell that releases a pro-inflammatory cytokine when exposed to a vertebrate HMGB B box or biologically active fragment thereof, and (b) a compound combined with the HMGB B box or biologically active fragment thereof. And then examining whether the compound inhibits the release of proinflammatory cytokines from the cell when compared to an appropriate control. Compounds that inhibit the release of proinflammatory cytokines in this assay are compounds that can be used to treat inflammation and / or inflammatory responses. The HMGB B box or biologically active HMGB B box fragment can be endogenous to the cell or can be introduced into the cell using standard recombinant molecular biology techniques.

  Any cell that releases pro-inflammatory cytokines in response to exposure to a vertebrate HMGB B box or biologically active fragment thereof in the absence of the test compound is expected to be useful in the present invention. The selected cells are expected to be important in the pathogenesis of the condition to be treated with the inhibitory compound being tested. For many conditions, the preferred cell is expected to be a human macrophage.

  Any method that tests whether a compound inhibits the release of pro-inflammatory cytokines from cells would be useful in these embodiments. A preferred method is expected to be a direct measurement of pro-inflammatory cytokines using, for example, any of a number of commercially available ELISA assays. However, in some embodiments, measurement of the inflammatory effect of released cytokines may be preferred, particularly when there are several pro-inflammatory cytokines produced by the test cell. As mentioned above, in many important disorders, the predominant pro-inflammatory cytokine is TNF, IL-1α, IL-1β, MIF or IL-6, in particular TNF.

  The invention also features a method for determining whether a compound increases an inflammatory response and / or inflammation. The method comprises (a) a cell that releases a pro-inflammatory cytokine when exposed to a vertebrate HMGB A box or biologically active fragment thereof, and (b) a compound (test compound) in the HMGB A box or biologically active fragment. ), And then examining whether the compound increases the release of pro-inflammatory cytokines from the cell relative to the appropriate control. A compound that increases the release of pro-inflammatory cytokines in this assay is a compound that can be used to increase the inflammatory response and / or inflammation. The HMGB A box or HMGB A box biologically active fragment can be endogenous to the cell or can be introduced into the cell using standard recombinant molecular biological techniques.

  Proinflammatory cytokines in response to exposure to vertebrate HMGB A boxes or biologically active fragments thereof in the absence of any test compound as well as cell types useful for the identification of inhibitors of inflammation as described above Any cell that normally suppresses the release of would be expected to be useful in the present invention. It is expected that the selected cells will be important in the pathogenesis of the condition to be treated with the inhibitory compound being tested. For many conditions, the preferred cell is expected to be a human macrophage.

  Any method that tests whether a compound increases the release of pro-inflammatory cytokines from cells would be useful in these embodiments. A preferred method is expected to be a direct measurement of pro-inflammatory cytokines using, for example, any of a number of commercially available ELISA assays. However, in some embodiments, measurement of the inflammatory effect of released cytokines may be preferred, particularly when there are several pro-inflammatory cytokines produced by the test cell. As mentioned above, in many important disorders, the predominant pro-inflammatory cytokine is TNF, IL-1α, IL-1β, MIF or IL-6, in particular TNF.

  Preferred embodiments of the invention are illustrated by the following examples. Other embodiments within the scope of the invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention as disclosed herein. The specification is intended to be considered exemplary only with examples and claims.

Example 1
Materials and methods
Cloning of HMGB1 and production of HMGB1 mutant Human HMGB1 clones and mutants were prepared using the following method. Using the following primers: forward primer: 5′GATGGGCAAAGGAGATCCTAAG3 ′ (SEQ ID NO: 6) and reverse primer: 5′GCGGCCGCTTATTCATCATCATCATCTTCTC3 ′ (SEQ ID NO: 7), a human brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Recombinant full-length human HMGB1 (651 base pairs; GenBank accession number U51677) was cloned from CA) by PCR amplification. Human HMGB1 mutant was cloned and purified as follows. The truncated form of human HMGB1 was cloned by PCR amplification from a human brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.). The primers used are as follows (forward and reverse respectively).
Carboxy terminal mutant (557 bp): 5′GATGGGCAAAGGAGATCCTAAG3 ′ (SEQ ID NO: 8) and 5′GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC3 ′ (SEQ ID NO: 9).
Amino terminus + B box mutant (486 bp): 5'GAGCATAAGAAGAAGCACCCA3 '(SEQ ID NO: 10) and 5'GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC3' (SEQ ID NO: 11).
B box mutant (233 bp): 5′AAGTTCAAGGATCCCAATGCAAAG3 ′ (SEQ ID NO: 12) and 5′GCGGCCGCTCAATATGCAGCTATATCCTTTTC3 ′ (SEQ ID NO: 13).
Amino terminus + A box mutant (261 bp): 5′GATGGGCAAAGGAGATCCTAAG3 ′ (SEQ ID NO: 14) and 5′TCACTTTTTTGTCTCCCCTTTGGG3 ′ (SEQ ID NO: 15).

  Protein size accuracy was ensured by adding a stop codon to each mutant. The PCR product was subcloned into the pCRII-TOPO vector EcoRI site using the TA cloning method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Following amplification, the PCR product was digested with EcoRI and subcloned into the expression vector with GST tag pGEX (Pharmacia); the correct orientation and positive clones were confirmed by DNA sequencing on both strands. Recombinant plasmids were transformed into protease-deficient E. coli strains BL21 or BL21 (DE3) plysS (Novagen, Madison, Wis.) And fusion protein expression was induced with isopropyl-D-thiogalactopyranoside (IPTG). The recombinant protein was obtained using affinity purification using a glutathione sepharose resin column (Pharmacia).

The HMGB variant prepared as described above has the following amino acid sequence:
Wild type HMGB1:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKT
MSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAF
FLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKE
KYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEEEDEE
DEEDEEEDDDDE (SEQ ID NO: 18)

Carboxy terminal mutant:
MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKT
MSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAF
FLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKE
KYEKDIAAYRAKGKPDAAKKGVVKAEKSK (SEQ ID NO: 19)

B box mutant: FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEM
WNNTAADDKQPYEKKAAKLKEKYEKDIAAY (SEQ ID NO: 20)

Amino terminal + A box mutant: MGKGDPKKPTGKMSSYAFFVQTCREEHKKK
HPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPP
KGET (SEQ ID NO: 21), where A box is an array
PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK
It consists of FEDMAKADKARYEREMKTYIPPKGET (SEQ ID NO: 22).

  Polypeptides made from GST vectors lacking HMGB1 protein were included as controls (containing only GST tags). DNaseI (Life Technologies) for carboxy-terminal and B-box mutants, or wild-type HMGB1 to inactivate bacterial DNA bound to wild-type HMGB1 and some mutants (carboxy-terminal and B-box) Benzonase nuclease (Novagen, Madison, Wis.) Was added at approximately 20 units / ml bacterial lysate. DNA degradation was confirmed by ethidium bromide staining of agarose gel containing HMGB1 protein before and after treatment. The protein eluate was passed through a polymyxin B column (Pierce, Rockford, IL) to remove the existing LPS and dialyzed thoroughly against phosphate buffered saline to remove excess reduced glutathione. The preparation was then lyophilized and redissolved in sterile water before use. LPS concentrations were less than 60 pg / μg protein for all mutants and 300 pg / μg for wild-type HMG-1 as determined by the Limulus amoeba-like cell degradation product test (Bio Whittaker Inc., Walkersville, MD). The integrity of the protein was confirmed using SDS-PAGE. Recombinant rat HMGB1 (Wang et al., Science 285: 248-251, 1999) was used in some experiments because it does not have a degradation fragment as observed with purified human HMGB1.

Peptide synthesis Peptides were synthesized at 90% purity at the Utah State University Biotechnology Center (Logan, Utah) and purified by HPLC. Endotoxin could not be detected in synthetic peptide preparations as determined by the Limulus assay.

Cell culture Mouse macrophage-like RAW264.7 cells (American Type Culture Collection, Rockville, MD) in RPMI1640 medium (Life Technologies, Grand) supplemented with 10% fetal calf serum (Gemini, Catabasas, CA), penicillin and streptomycin (Life Technologies) Island NY) and 90% confluent in serum-free Opti-MEM I medium (Life Technologies, Grand Island, NY). Polymyxin B (Sigma, St. Louis, MO) is routinely added at 100-1,000 units / ml to neutralize any mixed LPS activity as described above; only polymyxin B is trypan blue (Wang et al., It did not affect the viability of the cells evaluated in the above. Polymyxin B was not used in synthetic peptide research experiments.

Measurement of TNF release from cells
TNF release was determined by standard mouse fibroblast L929 (ATCC, American Type Culture Collection, Rockville, MD) cytotoxicity bioassay (Bianchi et al., Journal of Experimental Medicine 183: 927-936, 1996) with a minimum detectable concentration of 30 pg / Measured in ml. Recombinant mouse TNF was obtained from R & D system Inc. (Minneapolis, Minn.). Mouse fibroblast L929 cells (ATCC) in 10% fetal bovine serum (Gemini, Catabasas, CA), penicillin (50 units / ml) and streptomycin (50 μg / ml) in a 5% CO ₂ humidified incubator (Life Technologies) In DMEM (Life Technologies, Grand Island, NY) supplemented with

Antibody production
Polyclonal antibodies against the HMGB1 B box were generated in rabbits (Cocalico Biologicals, Inc., Reamstown, Pa.) And assayed for titer by immunoblotting. IgG was purified from anti-HMGB1 antiserum using protein A agarose according to manufacturer's instructions (Pierce, Rockford, IL). Anti-HMGB1 B box antibody was affinity purified using cyanogen bromide activated Sepharose beads (Cocalico Biological, Inc.). Non-immune rabbit IgG was purchased from Sigma (St. Louis, MO). The antibody detected full length HMGB1 and B box in an immunoassay but did not cross-react with TNF, IL-1 and IL-6.

Labeling and cell surface binding of HMGB1 with Na- ¹²⁵ I Purified HMGB1 protein (10 μg) using Iodo-beads (Pierce, Rockford, IL) without carrier ¹²⁵ I (NEN Life Science Products Inc., Boston, MA) radiolabeled with 0.2 mCi. A gel chromatography column (P6 Micro Bio-) in which ¹²⁵ I-HMGB1 protein was pre-equilibrated with unreacted ¹²⁵ I to 300 mM sodium chloride, 17.5 mM sodium citrate, pH 7.0 and 0.1% bovine serum albumin (BSA). Spin chromatography column, Bio-Rad Laboratories, Hercules, CA). The specific activity of the eluted HMGB1 was about 2.8 × 10 ⁶ cpm / μg protein. Cell surface binding studies were performed as previously described (Yang et al., Am. J. Physiol. 275: C675-C683, 1998). RAW264.7 cells were plated in 24-well dishes and grown to confluence. Cells were washed twice with ice-cold PBS containing 0.1% BSA, binding was 120 mM sodium chloride, 1.2 mM magnesium sulfate, 15 mM sodium acetate, 5 mM potassium chloride, 10 mM Tris.HCl, pH 7.4, 0.2% BSA, 5 mM glucose and 25,000 The reaction was performed at 4 ° C. for 2 hours using 0.5 ml of a binding buffer containing cpm ¹²⁵ I-HMGB1. At the end of the incubation, the supernatant was discarded and the cells were washed 3 times with 0.5 ml ice-cold PBS containing 0.1% BSA and lysed with 0.5 ml 0.5 N NaOH and 0.1% SDS for 20 minutes at room temperature. The radioactivity in the lysate was then measured using a gamma counter. Specific binding was determined as the radioactivity obtained in the presence of excess amounts of unlabeled HMGB1 or A box protein minus total binding.

Animal experimentation
TNF knockout mice were obtained from Amgen (Thousand Oaks, CA) and were on a B6x129 background. Age-matched wild-type B6x129 mice were used as a study control. Mice were bred within the population at the University of Florida specific pathogen-free transgenic mouse facility (Gainesville, FL) and used at 6-8 weeks of age.

  Male 6-8 week old Balb / c and C3H / HeJ mice were purchased from Harlen Sprague-Dawley (Indianapolis, IN) and acclimated for 7 days prior to use in experiments. All animals were housed under standard temperature and light / dark cycles at North Shore University Hospital Animal Facility.

Cecal ligation and puncture Cecal ligation and puncture (CLP) was performed as previously described (Fink and Hard, J. Surg. Res. 49: 186-196, 1990; Wichmann et al., Crit. Care Med. 26: 2078-2086, 1998; and Remick et al., Shock 4: 89-95, 1995). Briefly, Balb / c mice were anesthetized with 75 mg / kg ketamine (Fort Dodge, Fort Dodge, Iowa) and 20 mg / kg xylazine (Bohringer Ingelheim, St. Joseph, MO) administered intramuscularly. A midline incision was made and the cecum was isolated. A 6-0 prolene suture ligature was placed at a depth of 5.0 mm from the cecal tip in a direction away from the ileocecal valve.

  The ligated cecal stump was then punctured once using a 22 gauge needle without draining feces directly. The cecum was then returned to the normal intraperitoneal position. Next, in order to prevent fluid leakage, the peritoneum and fascia were separated into two layers and the abdomen was closed with 6-0 prolene running suture. All animals were resuscitated by subcutaneous administration of normal saline at 20 ml / kg body weight. Each mouse was subcutaneously injected with imipenem (0.5 mg / mouse) (Primaxin, Merck & Co., Inc., West Point, PA) 30 minutes after the operation. The animals were then recovered. Mortality was recorded up to 1 week after treatment, and surviving animals were followed up for 2 weeks to confirm that there was no subsequent death.

D-galactosamine sensitized mouse
D-galactosamine sensitization models have been previously reported (Galanos et al., Proc Natl. Acad. Sci. USA 76: 5939-5943, 1979; and Lehmann et al., J. Exp. Med. 165: 657-663, 1997. ). Mice were injected intraperitoneally with 20 mg D-galactosamine-HCL (Sigma) / mouse (in 200 μl PBS) and either 0.1 or 1 mg HMGB1 B box or vector protein (in 200 μl PBS). Daily deaths were recorded up to 72 hours after injection, surviving animals were followed for 2 weeks, and no subsequent deaths due to B box toxicity were observed.

Spleen bacterial cultures 14 mice were given either anti-HMGB1 antibody (n = 7) or control (n = 7) at 24 and 30 hours after CLP as described herein, euthanized and subjected to necropsy did. Spleen bacteria were harvested as previously described (Villa et al., J. Endotoxin Res. 4: 197-204, 1997). The spleen was removed by aseptic technique and homogenized in 2 ml PBS. After serial dilution in PBS, homogenates were plated 0.15 ml on trypsin soy agar plates (Difco, Detroit, MI) and incubated overnight at 37 ° C. before counting CFU.

Statistical analysis Data were expressed as mean ± SEM unless otherwise noted. Differences between groups were examined by two-tailed Student's t-test, one-way ANOVA, followed by least significant difference test or two-tailed Fisher exact test.

Example 2
Mapping of HMGB1 domain for induction of cytokine activity
HMGB1 has two DNA binding domains (A and B boxes) and a negatively charged acidic carboxyl tail (tail). To elucidate the structural basis of HMGB1 cytokine activity and to map inflammatory protein domains, we expressed the full length and truncation form of HMGB1 by mutagenesis, and for stimulatory activity in monocyte culture The purified protein was screened (Figure 1). A full length HMGB1, a mutant lacking the carboxy terminus, a mutant containing only the B box and a mutant having only the A box were made. These variants of human HMGB1 are generated by polymerase chain reaction (PCR) using the specific primers described herein, and the mutant protein is a glutathione S-transferase (GST) gene according to the manufacturer's instructions. It was expressed using a fusion system (Pharmacia Biotech, Piscataway, NJ). In other words, a DNA fragment prepared by PCR was fused to a GST fusion vector and amplified in E. coli. The expressed HMGB1 protein and HMGB1 mutant were then isolated using a GST affinity column.

  The effect of the mutant on the release of TNF from mouse macrophage-like RAW264.7 cells (ATCC) was performed as follows. RAW264.7 cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island NY) supplemented with 10% fetal calf serum (Gemini, Catabasas, CA), penicillin and streptomycin (Life Technologies). Polymyxin (Sigma, St. Louis, MO) was added at 100 units / ml to suppress any mixed LPS activity. Cells were incubated for 8 hours in Opti-MEM I medium with 1 μg / ml full length (wild type) HMGB1 and each HMGB1 mutein. Conditioned supernatant (containing TNF released from cells) is collected and TNF released from cells is collected by standard mouse fibroblast L929 (ATCC) cytotoxicity bioassay (Bianchi et al., Supra). The minimum detection concentration was 30 pg / ml. Recombinant mouse TNF was obtained from R & D Systems Inc. (Minneapolis, Minn.) And used as a control in these experiments. The results of this study are shown in FIG. All data in FIG. 1 were expressed as mean + SEM unless otherwise specified (N = 6-10).

  As shown in FIG. 1, wild type HMGB1 and carboxyl truncation HMGB1 significantly stimulated TNF release by monocyte cultures (mouse macrophage-like RAW264.7 cells). B box was a strong activator of monocyte TNF release. The stimulatory effect of this B box was specific because the A box only activated TNF release only slightly.

Example 3
HMGB1 B box protein promotes cytokine activity in a dose-dependent manner
To further investigate the effect of HMGB1 B box on cytokine production, the effect of various amounts of HMGB1 B box on TNF, IL-1B and IL-6 production in mouse macrophage-like RAW264.7 cells was evaluated. RAW264.7 cells were stimulated with 0-10 μg / ml of B box protein for 8 hours as shown in FIGS. Conditioned medium was collected and the concentrations of TNF, IL-1β and IL-6 were measured. The concentration of TNF is measured as described herein, and the concentration of mouse IL-1β and IL-6 is determined by enzyme-linked immunosorbent assay (ELISA) kit of mouse IL-1β and IL-6 (R & D System Inc. Minneapolis , MN) and N> 5 for all experiments. The results of the test are shown in FIGS.

  As shown in FIG. 2A, TNF release from RAW264.7 cells increased as the amount of B box administered to the cells increased. As shown in FIG. 2B, the addition of 1 μg / ml or 10 μg / ml B box increased IL-1β release from RAW264.7 cells. Furthermore, as shown in FIG. 2C, IL-6 release from RAW264.7 cells increased as the amount of B box administered to the cells increased.

  The kinetics of B box induced TNF release was also investigated. TNF release and TNF mRNA expression were measured in RAW264.7 cells induced 0-48 hours with B-box polypeptide or GST-tag polypeptide alone (10 μg / ml) used as a control (vector). The supernatant TNF protein concentration was analyzed by the L929 cytotoxicity test (N = 3-5) as described herein. For mRNA measurements, cells were plated on 100 mm plates and treated for 0, 4, 8 or 24 hours in Opti-MEM I medium containing only B box polypeptide or vector as shown in FIG. 2D. Vector-only samples were tested at 4 hours. Cells were scraped from the plate and total RNA was isolated using the RNAzolB method according to the manufacturer's instructions (Tel-Test “B”, Inc., Friendswood, TX). TNF (287 bp) was measured by RNase protection test (Ambion, Austin, TX). Equal loading and RNA integrity were confirmed by ethidium bromide staining of RNA samples on agarose formaldehyde gel. The results of the RNase protection test are shown in FIG. 2D. As shown in FIG. 2D, monocyte B box activation occurred at the level of gene transcription, as TNF mRNA was significantly increased in monocytes exposed to B box protein (FIG. 2B). TNF mRNA expression was highest at 4 hours and decreased at 8 and 24 hours. The vector only control (GST tag) had no effect on TNF mRNA expression. A TNF protein from RAW264.7 cells 0, 4, 8, 24, 32, or 48 hours after administration of the B box or vector alone (GST tag) using the L929 cytotoxicity test described herein for a similar study Performed while measuring release. Compared to the control (medium only), B box treatment stimulated TNF protein expression (FIG. 2E), and only the vector did not (FIG. 2F). Data represent three separate experiments. Both these data indicate that the HMGB1 B box domain has cytokine activity and is responsible for the cytokine-stimulating activity of full-length HMGB1.

  In summary, the HMGB1 B box stimulated the release of TNF, IL-1β and IL-6 from monocyte cultures in a dose-dependent manner (FIGS. 2A-2C), consistent with the inflammatory activity of full-length HMGB1 ( Andersson et al., J. Exp. Med. 192: 565-570, 2000). Furthermore, these studies indicate that maximum TNF protein release occurs within 8 hours (FIG. 2E). This delayed pattern of TNF release is similar to the TNF release induced by HMGB1 itself, and is significantly delayed than the TNF kinetics induced by LPS (Andersson et al., Supra).

Example 4
The first 20 amino acids of the HMGB1 B box stimulate TNF activity
The TNF stimulating activity of HMGB1 B box was further mapped. This study was conducted as follows. The B box fragment was generated using synthetic peptide protection methods as described herein. Five HMGB1 B box fragments (derived from SEQ ID NO: 20) containing amino acids 1-20, 16-25, 30-49, 45-64 or 60-74 of the HMGB1 B box were generated as shown in FIG. RAW264.7 cells were treated with B box (1 μg / ml) or a synthetic peptide fragment of B box (10 μg / ml) as shown in FIG. 3 for 10 hours, and the release of TNF in the supernatant is described herein. Measured as described. Data shown are mean ± SEM (n = 3 experiments, each performed in duplicate, confirmed using 3 different lots of synthetic peptides). As shown in FIG. 3, the TNF stimulating activity was retained by a synthetic peptide corresponding to amino acids 1 to 20 (fkdpnapkrlpsafflfcse; SEQ ID NO: 23) of the HMGB1 B box of SEQ ID NO: 20. The 1-20mer TNF stimulating activity was less intense than either the full-length synthetic B box (1-74mer) or the full-length HMGB1, but the stimulatory effect was 16-25, 30-49, 45-64 of the HMGB1 B box or Synthesis of amino acid fragments containing 60-74 was specific because it did not induce TNF release. These results are direct evidence that the B box macrophage stimulating activity is specifically located at the first 20 amino acids of the HMGB B box domain of SEQ ID NO: 20). This B-box fragment is a polypeptide encoding a full-length B-box polypeptide, e.g. for stimulating the release of pro-inflammatory cytokines or for treating conditions in patients characterized by activation of the inflammatory cytokine cascade. It can be used in a similar manner.

Example 5
HMGB1 A box protein antagonizes HMGB1-induced cytokine activity in a dose-dependent manner A weak agonist is an antagonist by definition. As shown in FIG. 1, since the HMGB1 A box only weakly induces TNF production, the ability of the HMGB1 A box to act as an antagonist of HMGB1 activity was evaluated. This study was conducted as follows. Subconfluent RAW264.7 cells in 24-well dishes were treated with HMGB1 (1 μg / ml) and 0, 5, 10, or 25 μg / ml A box for 16 hours in Opti-MEM I medium with polymyxin B (100 units / unit). ml). TNF-stimulating activity (tested using the L929 cytotoxicity test described herein) in samples not dosed with A box was taken as 100%, and A box inhibition was expressed as a percentage of HMGB1 alone. The results of the A box effect on TNF release from RAW264.7 cells are shown in FIG. 4A. As shown in FIG. 4A, the A box suppressed HMGB1-induced TNF release in a dose-dependent manner, with an apparent EC ₅₀ value of approximately 7.5 μg / ml. Data in FIG. 4A are shown as mean ± SD (n = 2-3 independent experiments).

Example 6
HMGB1 A box protein suppresses cytokine activity of full-length HMGB1 and HMGB1 B boxes
Antagonism of full-length HMGB1 activity by HMGB1 A-box or GST tag (vector control) was also examined by measuring TNF release from RAW264.7 macrophage cultures stimulated by co-addition of A-box with full-length HMGB1. RAW264.7 macrophage cells (ATCC) were seeded in 24-well tissue culture plates and used at 90% confluence. Cells in HMGB1 and / or A box as described for 16 hours in Optimum I medium (Life Technologies, Grand Island, NY) in the presence of polymyxin B (100 units / ml, Sigma, St. Louis, MO) The supernatant was collected and subjected to TNF measurement (from mouse ELISA kit, R & D System Inc, Minneapolis, Minn.). TNF-inducing activity was expressed as the percentage of activity achieved with HMGB1 alone. The results of these studies are shown in FIG. 4B. FIG. 4B is a histogram of the effects of HMGB1 (HMG-1) alone, A box alone, vector (control) alone, HMGB1 and A box combination, and HMGB1 and vector combination. As clearly shown in FIG. 4B, the HMGB1 A box significantly attenuated the TNF-stimulating activity of full-length HMGB1.

Example 7
HMGB1 A box protein suppresses HMGB1 cytokine activity by binding to it
To examine whether the HMGB1 A box functions as an antagonist by replacing HMGB1 binding, ¹²⁵ I-labeled HMGB1 was added to macrophage cultures and binding was measured after 2 hours at 4 ° C. Binding assays in RAW264.7 cells were performed as described herein. ¹²⁵ I-HMGB1 binding was measured in RAW264.7 cells plated in time 24-well dishes as shown in FIG. 5A. The specific binding described is equivalent to the total cell bound ¹²⁵ I-HMGB1 (CPM / well) minus the cell bound CPM / well in the presence of a 5000-fold molar excess of unlabeled HMGB1. FIG. 5A is a graph of ¹²⁵ I-HMGB1 binding over time. As shown in FIG. 5A, HMGB1 exhibited saturable primary binding kinetics. Specificity of binding was assessed as described in Example 1.

In addition, ¹²⁵ I-HMG-1 binding was measured in RAW264.7 cells plated in 24-well dishes and incubated in the presence of ¹²⁵ I HMGB1 alone or unlabeled HMGB1 or A box. The results of this binding assay are shown in FIG. 5B. Data represent the mean ± SEM of three individual experiments. FIG. 5B shows ¹²⁵ I-HMGB1 in the absence of unlabeled HMGB1 or HMGB1 A box or in the presence of a 5000 molar excess of unlabeled HMGB1 or HMGB1 A box, measured as a percentage of total CPM / well. It is a histogram of cell surface binding. In FIG. 5B, “total” is equal to counts per minute (CPM) / well of cell-bound ¹²⁵ I-HMGB1 in the absence of unlabeled HMGB1 or A box for 2 hours at 4 ° C. “HMGB1” or “A box” is equivalent to CPM / well of cell-bound ¹²⁵ I-HMGB1 in the presence of a 5000 molar excess of unlabeled HMGB1 or unlabeled A box. Data are expressed as a percentage of the total count (2,382,179 CPM / well) obtained in the absence of unlabeled HMGB1 protein. These results indicate that the HMGB1 A box is a competitive antagonist of HMGB1 activity in vitro and suppresses TNF-stimulating activity of HMGB1.

Example 8
Inhibition of full length HMGB1 and HMGB1 B box cytokine activity by anti-B box polyclonal antibody.
The ability of antibodies directed against the HMGB1 B box to modulate full-length or HMGB1 B box action was also evaluated. An affinity purified antibody directed against the HMGB1 B box (B box antibody) was generated as described herein and using standard techniques. To test the effect of antibodies on HMGB1-induced or HMGB1 B-box induced TNF release from RAW264.7 cells, subconfluent RAW264.7 cells in 24-well dishes were treated with anti-B box antibody (25 μg / ml or 100 μg / ml antigen). Affinity purification, Cocalico Biologicals, Inc., Reamstown, PA) or HMG-1 (1 μg / ml) or HMGB1 B box (with or without addition of non-immune IgG (25 μg / ml or 100 μg / ml; Sigma) for 10 hours 10 μg / ml). TNF release from RAW264.7 cells was measured using the L929 cytotoxicity test as described herein. The results of this study are as shown in FIG. 6, which shows no administration, 1 μg / ml HMGB1, 1 μg / ml HMGB1 + 25 μg / ml anti-B box antibody, 1 μg / ml HMGB1 + 25 μg / ml IgG ( Control), released by RAW264.7 cells administered 10 μg / ml B box, 10 μg / ml B box + 100 μg / ml anti-B box antibody or 10 μg / ml B box + 100 μg / ml IgG (control) It is a histogram of TNF performed. The amount of TNF released from cells induced by HMGB1 alone (no B box antibody added) was taken as 100%, and the data shown in FIG. 6 are the results of three independent experiments. As shown in FIG. 6, affinity purified antibodies directed against the HMGB1 B box significantly inhibited TNF release induced by either full-length HMGB1 or HMGB1 B box. These results indicate that such antibodies can be used to modulate the function of HMGB1.

Example 9
HMGB1 B box protein is toxic to D-galactosamine sensitized Balb / c mice
To investigate whether the HMGB1 B box has cytokine activity in vivo, we were sensitized with D-galactosamine (D-gal), a model that has been widely used to study cytokine toxicity. HMGB1 B box protein was administered to unanesthetized Balb / c mice (Galanos et al., Supra). In other words, as shown in Table 1, mice (20-25 grams, male, Harlan Sprague-Dawley, Indianapolis, IN) to D-gal (20 mg) (Sigma, St. Louis, Missouri) and B box (0.1 mg / ml / mouse or 1 mg / ml / mouse) or GST tag (vector, 0.1 mg / ml / mouse or 1 mg / ml / mouse) was injected intraperitoneally. Survival of mice was monitored up to 7 days to confirm that there were no late deaths. The results of this test are shown in Table 1.

  The results of this study showed that the HMGB1 B box is lethal to D-galactosamine sensitized mice in a dose-dependent manner. In all cases where death occurred, it had occurred within 12 hours. No lethality was observed in mice treated with a corresponding preparation of purified GST vector protein without the B box.

Example 10
Histological characteristics of D-galactosamine-sensitized Balb / c mice or C3H / Hej mice treated with HMGB1 B box protein To further evaluate the lethality of HMGB1 B box protein in vivo, HMGB1 B box was re-D- Galactosamine-sensitized Balb / c mice were administered. Mice (3 per group) were given D-gal (20 mg / mouse) + B box or vector (1 mg / mouse) intraperitoneally for 7 hours, then decapitated and sacrificed. Blood was collected and organs (liver, heart, kidney and lung) were collected and fixed in 10% formaldehyde. Tissue sections were prepared by hematoxylin and eosin staining and subjected to histological evaluation (Criterion Inc., Vancouver, Canada). The results of these tests are shown in FIGS. 7A-7J, which show kidney sections (FIG. 7A), myocardial sections (FIG. 7C), lung sections (FIG. 7E) and liver sections (FIG. 7) obtained from untreated mice stained with hematoxylin and eosin. 7G and 7I), and scanned images of kidney sections (Figure 7B), myocardial sections (Figure 7D), lung sections (Figure 7F) and liver sections (Figures 7H and 7J) obtained from mice treated with HMGB1 B box . Compared to control mice, B box treatment did not cause abnormalities in the kidneys (Figures 7A and 7B) and lungs (Figures 7E and 7F). The mice showed some ischemic changes and disappearance of the cross-striatal of cardiac myocardial fibers (shown by arrows in FIGS. 7C and 7D, FIG. 7D). The liver showed the majority of B box damage as shown by ongoing hepatitis (FIGS. 7G-7J). In FIG. 7J, hepatocyte shedding is observed surrounded by accumulated polymorphonuclear leukocytes. The arrows in FIG. 7J indicate the site of polymorphonuclear accumulation (dotted line) or apoptotic hepatocytes (solid line). In vivo administration of HMGB1 B box also significantly increased serum concentrations of IL-6 (315 + 93 vs 20 + 7 pg / ml, B box vs control, p <0.05) and IL-1β (15 + 3 vs 4 +1 pg / ml, B box versus control, p <0.05).

  Administration of B box protein to C3H / HeJ mice (not responsive to endotoxin) is also lethal, indicating that the HMGB1 B box is lethal in the absence of LPS signaling. Lung and kidney hematoxylin and eosin-stained sections taken 8 hours after administration of B box showed no abnormal morphological changes. However, examination of sections obtained from the heart revealed signs of ischemia with the disappearance of the cross streak with irregular pink cytoplasm in the myocardial fibers. Liver sections showed some acute hepatocyte shedding and apoptosis, and a mild acute inflammatory response with sporadic polymorphonuclear leukocytes. These specific pathological changes are comparable to those observed after administration of full-length HMGB1, confirming that the B box alone can repeat a lethal pathological response to HMGB1 in vivo.

  In order to examine whether the TNF-stimulating activity of HMGB1 contributes to lethality mediation by B box, the present inventors sensitized with D-glucosamine (20 mg / mouse), and B box (1 mg / mouse, intraperitoneal). Injection) exposed to TNF knockout mice (TNF-KO, Nowak et al., Am.J.Physiol.Regul.Integr.Comp.Physiol.278: R1202-R1209,2000) and wild type controls (B6x129 strain). It was measured. The B box was highly lethal (6 of 9 exposed) to wild type mice, but lethality was not observed in TNF-KO mice treated with B box (exposed) 0 of 9 animals died, p <0.05 vs (v. Wild type). Together with the RAW264.7 macrophage culture data described herein, these data now indicate that the HMGB1 B box confers specific TNF-stimulated cytokine activity.

Example 11
HMGB1 protein concentration increases in septic mice To investigate the role of HMGB1 in sepsis, we established sepsis in mice and measured serum HMGB1 using previously described quantitative immunoassays (Wang Et al., Supra). Mice were subjected to cecal ligation and perforation (CLP), a well-characterized model of sepsis generated by puncturing a surgically created cecal diverticulum leading to polymicrobial peritonitis and sepsis (Fink and Head, Supra; Wichmann et al., Supra; and Remick et al., Supra). Next, the serum concentration of HMGB1 was measured (Wang et al., Supra). FIG. 8 shows the results of this study in a graph showing the concentration of HMGB1 in mice after 0, 8, 18, 24, 48 and 72 hours subjected to CLP. As shown in FIG. 8, the serum HMGB1 concentration did not increase significantly during the first 8 hours after cecal puncture, and then increased significantly after 18 hours (FIG. 8). Elevated serum HMGB1 maintains an elevated plateau state for at least 72 hours after CLP, and its kinetic characteristics are very similar to previously reported delayed HMGB1 kinetics in endotoxemia (Wang et al., Previous Out). This transient pattern of HMGB1 release closely corresponded to the onset of sepsis signs in mice. During the first 8 hours after cecal puncture, the animals showed a mild disease state, some with reduced motility and decreased exploratory behavior. Over the next 18 hours, the animals were in a severe condition, gathered in a raised state, did not search for water and samples, and responded little to external stimuli and breeder tests.

Example 12
Treatment of sepsis mice with HMGB1 A box protein increases mouse viability.
In order to investigate whether the HMGB1 A box can suppress the lethality of HMGB1 during sepsis, mice were subjected to cecal puncture and treated by administration of A box from 24 hours after onset of sepsis. CLP was performed on male Balb / c mice as described herein. Animals were randomly grouped to have 15-25 mice per group. HMGB1 A box (60 or 600 μg / mouse, each time) or vector (GST tag, 600 μg / mouse) alone was intraperitoneally administered twice a day for 3 days starting from 24 hours after CLP. Survival was monitored twice a day for up to 2 weeks to confirm that there were no late deaths. The results of this study are shown in FIG. 9, which is a graph of the effect of vector (GST; control) 60 μg / mouse or 600 μg / mouse on survival over time (* P <0.03 vs. control, Fisher's exact test) by). As shown in FIG. 9, administration of HMGB1 A box significantly rescued mice from the lethal effects of sepsis and from 28% in animals treated with proteins purified from vector protein (GST) containing no A box. Survival improved to 68% in boxed animals (P <0.03, according to Fisher's exact test). The rescue action of HMGB1 A box in this sepsis model depends on the A box dose; animals treated with A box 600 μg / mouse were treated with control animals treated with vector-derived preparations or with only 60 μg A box Compared with any of the animals, the animals showed significantly higher alertness, motility, and recovered feeding behavior. The latter animals remained in a severe condition, continued with reduced activity and feeding behavior for several days, and most died.

Example 13
Treatment of sepsis mice with anti-HMGB1 antibody increases mouse viability. Passive immunization with anti-HMGB1 antibody in sepsis mice, which is a serious disease state, was also evaluated. In this study, male Balb / c mice (20-25 gm) were subjected to CLP as described herein. Affinity-purified anti-HMGB1 B box polyclonal antibody or rabbit IgG (as a control) was administered 24 hours to 2 times a day for 3 days at 600 μg / mouse. Viability was monitored for 2 weeks. The results of this study are shown in FIG. 10A, which is a graph of the viability of sepsis mice treated with either control or anti-HMGB1 antibodies. Results show that anti-HMGB1 antibody administered to mice 24 hours after the start of cecal puncture significantly rescued animals from death compared to non-immune antibody administration (p <0.02, according to Fisher's exact test) ) Is shown. Within 12 hours after administration of anti-HMGB1 antibody, the treated animals showed increased activity and responsiveness compared to non-immune antibody-treated controls. Animals treated with non-immune antibodies gathered and remained poorly haired and less active, whereas treated animals improved significantly and returned to normal feeding behavior within 48 hours. In this model, anti-HMGB1 antibody is equivalent to the number of bacteria from the spleen 31 hours after CLP compared to animals to which the present inventors received an invalid antibody (CFU, aerobic colony forming unit). As a result, the growth of bacteria was not suppressed (the number of control bacteria = 3.5 ± 0.9 × 10 ⁴ CFU / g; n = 7). Animals were then monitored for up to 2 weeks, with no late deaths suggesting that treatment with anti-HMGB1 provided complete relief from lethal sepsis and did not only delay the time of death. It was.

  According to the knowledge of the present inventors, other specific cytokine-directed therapeutic agents are not so effective when administered so late after the onset of sepsis. By comparison, administration of anti-TNF actually increases mortality in this model, and anti-MIF antibodies are ineffective when administered more than 8 hours after cecal puncture (Remick et al., Supra; and Calandra et al., Nature Med. 6: 164-170, 2000). These data indicate that HMGB1 can be targeted as late as 24 hours after cecal puncture to rescue fatal cases of established sepsis.

  In another example of rescue of endotoxemic mice using anti-B box antibodies, the rescue ability of anti-HMGB1 B box antibodies in LPS-induced sepsis mice was evaluated. Male Balb / c mice (20-25 gm, 26 per group) were treated with an LD75 dose of LPS (15 mg / kg) by intraperitoneal injection (IP). Anti-HMGB1 B box or non-immune rabbit serum (0.3 ml per mouse each time, IP) was given at 0, +12 and +24 hours after LPS administration. Mouse viability was assessed over time. The results of this study are shown in FIG. 10B, which is a graph of the survival of sepsis mice that received anti-HMGB1 B box antibody or non-immune serum. As shown in FIG. 10B, the anti-HMGB1 B box antibody improved the survival of sepsis mice.

Example 14
Suppression of the HMGB1 signaling pathway using anti-RAGE antibodies Previous data implicated RAGE as an HMGB1 receptor capable of mediating neurite outgrowth during brain development and smooth muscle migration in wound healing (Hori et al., J Biol.chem. 270: 25752-25761,1995; Merenmies et al., J. Biol. Chem. 266: 16722-16729, 1991; and Degryse et al., J. Cell Biol. 152: 1197-1206, 2001). We have identified HMGB1 (1 μg / ml), LPS (0.1 μg / ml) or HMGB1 B box (1 μg / ml) in the presence of anti-RAGE antibody (25 μg / ml) or non-immune IgG (25 μg / ml). TNF release was measured in RAW264.7 cultures stimulated with. In other words, cells were seeded in 24-well tissue plates and used at 90% confluence. LPS (E. coli 0111: B4, Sigma, St. Louis, MO) was sonicated for 20 minutes before use. Cells were cultured in serum-free Opti-MEM I medium (Life Technologies) for 16 hours in the presence of anti-RAGE antibody (25 μg / ml) or non-immune IgG (25 μg / ml) as shown in FIG. 11A, HMGB1 (HMG-1; Treatment with 1 μg / ml), LPS (0.1 μg / ml) or HMGB1 B box (B box; 1 μg / ml), collecting supernatant and measuring TNF using the L929 cytotoxicity test described herein It was attached to. IgG purified polyclonal anti-RAGE antibody (Cat. No. sc-8230, N-16, Santa Cruz Biotech, Inc., Santa Cruz, CA) was dialyzed thoroughly against PBS before use. The results of this study are shown in FIG. 11A, which is a histogram of the effect of HMGB1, LPS or HMGB1 B box in the presence of anti-RAGE antibody or non-immune IgG (control) on TNF release from RAW264.7 cells . As shown in FIG. 11A, the anti-RAGE antibody significantly suppressed HMGB1 B box-induced TNF release compared to non-immune IgG. This inhibition was specific because anti-RAGE did not significantly inhibit LPS-stimulated TNF release. Importantly, the highest inhibitory action of anti-RAGE reduced HMG-1 signaling by only 40%, suggesting that other signaling pathways may be involved in HMGB1 signaling.

  In order to investigate the effect of HMGB1 or HMGB1 B box on NF-κB-dependent ELAM promoter, the following experiment was performed. As described in Means et al., (J. Immunol. 166: 4074-4082, 2001), RAW264.7 macrophages are mouse MyD88 dominant negative (DN) mutant (amino acids 146- Equivalent to 296) or transiently co-transfected with an empty vector plus a luciferase reporter plasmid. A portion of the cells was then stimulated with full length HMGB1 (100 ng / ml) or purified HMGB1 B box (10 μg / ml) for 5 hours. Cells were then harvested and luciferase activity was measured using standard methods. All transfections were performed in triplicate and repeated at least 3 times, and one representative experiment is shown in FIG. 11B. As shown in FIG. 11B, HMGB1 stimulated luciferase activity in samples that were MyD88 dominant negative and not co-transfected, and the level of stimulation was reduced in samples co-transfected with MyD88 dominant negative. This effect was also observed in samples administered HMGB B box.

  While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be appreciated by those skilled in the art that various changes in form and detail can be made without departing from the scope of the invention as set forth in the appended claims.

FIG. 1 is a schematic diagram showing the HMGB1 mutant and its activity in TNF release (pg / ml). FIG. 2A is a histogram showing the effect of B box 0 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml or 10 μg / ml on TNF release (pg / ml) in RAW264.7 cells. FIG. 2B is a histogram showing the effect of B box 0 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml or 10 μg / ml on IF-1β release (pg / ml) in RAW264.7 cells. FIG. 2C is a histogram showing the effect of B box 0 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml or 10 μg / ml on IL-6 release (pg / ml) in RAW264.7 cells. FIG. 2D is a scanning image of an RNAse protection test blot showing the effect of B box (0, 4, 8 or 24 hours after administration) or vector alone (4 hours after administration) on TNF mRNA expression in RAW264.7 cells. is there. FIG. 2E is a histogram of the effect of the HMGB1B box on TNF protein release (pg / ml) from RAW264.7 cells at 0, 4, 8, 24, 32 or 48 hours after administration. FIG. 2F is a histogram of the effect of the vector on TNF protein release (pg / ml) from RAW264.7 cells at 0, 4, 8, 24, 32 or 48 hours after administration. FIG. 3 is a schematic diagram showing the HMGB1B box mutant and its activity in TNF release (pg / ml). FIG. 4A is a graph of the effect of 0 μg / ml, 5 μg / ml, 10 μg / ml or 25 μg / ml of HMG1A box protein (as a percentage with HMGB1-mediated TNF release alone) on TNF release from RAW264.7 cells. . FIG. 4B shows the effect of HMGB1 (0 or 1.5 μg / ml), HMGB1 A box (0 or 10 μg / ml) or vector (0 or 10 μg / ml) alone or in combination on the release of TNF from RAW264.7 cells (HMGB1 Is a histogram) (as a percentage with only mediated TNF release). FIG. 5A is a graph of ¹²⁵ I-HMGB1 binding (CPM / well) binding to RAW264.7 cells over time (min). FIG. 5B shows the absence of unlabeled HMGB1 or HMGB1 A box as measured as a percentage of total CPM / well for 2 hours at 4 ° C. (total), or unlabeled HMGB1 (HMGB1) or A box (A box). 1 is a histogram of ¹²⁵ I-HMGB1 binding in the presence of a 5000 molar excess. FIG. 6 shows HMGB1 (HMG-1; 0 μg / ml or 1 μg / ml) or HMGB1B box (B box; 0 μg / ml or 10 μg / ml) alone or anti-B box antibody (for TNF release from RAW264.7 cells). Histogram of effects when combined with 25 μg / ml or 100 μg / ml) or IgG (25 μg / ml or 100 μg / ml) (expressed as a percentage for HMGB1-mediated TNF release only). FIG. 7A is a scanning image of a hematoxylin-eosin stained kidney section obtained from an untreated mouse. FIG. 7B is a scanning image of a hematoxylin eosin-stained kidney section obtained from an HMGB1B box-administered mouse. FIG. 7C is a scanning image of a hematoxylin-eosin stained myocardial section obtained from an untreated mouse. FIG. 7D is a scanning image of a hematoxylin eosin-stained myocardial section obtained from an HMGB1B box-administered mouse. FIG. 7E is a scanning image of a hematoxylin-eosin stained lung section obtained from an untreated mouse. FIG. 7F is a scanning image of a lung section stained with hematoxylin and eosin obtained from a mouse administered with HMGB1B box. FIG. 7G is a scanning image of a hematoxylin-eosin stained liver section obtained from an untreated mouse. FIG. 7H is a scanning image of a hematoxylin eosin-stained liver section obtained from a mouse administered with HMGB1B box. FIG. 7I is a scanning image (high magnification) of a hematoxylin-eosin stained liver section obtained from an untreated mouse. FIG. 7J is a scanning image (high magnification) of a hematoxylin eosin-stained liver section obtained from a mouse administered with HMGB1B box. FIG. 8 is a graph of HMGB1 concentration (ng / ml) over time (hours) in mice subjected to cecal ligation and puncture (CLP). FIG. 9 is a graph of the effect of HMGB A box (60 μg / mouse or 600 μg / mouse) or no administration on survival (day) of mice over time after cecal ligation and puncture (CLP). FIG. 10A is a graph of the effect of anti-HMGB1 antibody (black circles) or no treatment (white circles) on survival of mice over time (days) after cecal ligation and puncture (CLP). FIG. 10B is a graph of the effect of anti-HMGB1B box antiserum (black squares) or no administration (*) on the survival (days) of mice administered lipopolysaccharide (LPS). FIG. 11A is a histogram of the effect of anti-RAGE antibody or non-immune IGG on TNF release from RAW264.7 cells administered HMGB1 (HMG-1), lipopolysaccharide (LPS) or HMGB1B box (B box). FIG. 11B shows activation of NF-κB-dependent ELAM promoter (luciferase) in RAW264.7 cells co-transfected with mouse MyD88 dominant negative (+ myD88DN) mutation (corresponding to amino acids 146-296) or empty vector (-MyD88DN). 2 is a histogram of the effect of HMGB1 (HMG-1) or HMGB1B box (B box) polypeptide on (as measured by activity). Data are expressed as the ratio of mean luciferase values + SD (fold activation) obtained from unstimulated and stimulated cells (background subtraction). FIG. 12A is the amino acid sequence of human HMG1 polypeptide (SEQ ID NO: 1). FIG. 12B is the amino acid sequence of the rat and mouse HMG1 polypeptides (SEQ ID NO: 2). FIG. 12C is the amino acid sequence of human HMG2 (SEQ ID NO: 3). FIG. 12D is the amino acid sequence (SEQ ID NO: 4) of human, mouse and rat HMG1A box polypeptides. FIG. 12E is the amino acid sequence (SEQ ID NO: 5) of human, mouse and rat HMG1B box polypeptides. FIG. 12F is the nucleic acid sequence of the forward primer for human HMG1 (SEQ ID NO: 6). FIG. 12G is the nucleic acid sequence of the reverse primer for human HMG1 (SEQ ID NO: 7). FIG. 12H is the nucleic acid sequence (SEQ ID NO: 8) of the forward primer for the carboxy terminal mutant of human HMG1. FIG. 12I is the nucleic acid sequence of the reverse primer (SEQ ID NO: 9) for the carboxy terminal mutant of human HMG1. FIG. 12J is the nucleic acid sequence of the forward primer for the amino terminus + B box of human HMG1 (SEQ ID NO: 10). FIG. 12K is the nucleic acid sequence (SEQ ID NO: 11) of the reverse primer for the amino terminus + B box of human HMG1. FIG. 12L is the nucleic acid sequence (SEQ ID NO: 12) of the forward primer for the B box mutant of human HMG1. FIG. 12M is the nucleic acid sequence of the reverse primer for the B box mutant of human HMG1 (SEQ ID NO: 13). FIG. 12N is the nucleic acid sequence of the forward primer for the amino terminus + A box mutant of human HMG1 (SEQ ID NO: 14). FIG. 12O is the nucleic acid sequence of the reverse primer for the amino terminus + A box mutant of human HMG1 (SEQ ID NO: 15). FIG. 13 is a sequence alignment of rat (SEQ ID NO: 2), mouse (SEQ ID NO: 2) and human (SEQ ID NO: 18) HMGB1 polypeptide sequences. FIG. 14A is the nucleic acid sequence (SEQ ID NO: 32) of HMG1L5 (formerly HMG1L10) encoding HMGB polypeptide. FIG. 14B is the polypeptide sequence (SEQ ID NO: 24) of HMG1L5 (formerly HMG1L10) encoding HMGB polypeptide. FIG. 14C is the nucleic acid sequence of HMG1L1 (SEQ ID NO: 33) encoding HMGB polypeptide. FIG. 14D is the polypeptide sequence of HMG1L1 (SEQ ID NO: 25) encoding HMGB polypeptide. FIG. 14E is the nucleic acid sequence of HMG1L4 (SEQ ID NO: 34) encoding HMGB polypeptide. FIG. 14F is the polypeptide sequence of HMG1L4 (SEQ ID NO: 26) encoding HMGB polypeptide. FIG. 14G is the nucleic acid sequence (SEQ ID NO: 35) of the HMG polypeptide sequence of BAC clone RP11-395A23. FIG. 14H is the polypeptide sequence (SEQ ID NO: 27) of the HMG polypeptide sequence of BAC clone RP11-395A23 encoding HMGB polypeptide. FIG. 14I is the nucleic acid sequence of HMG1L9 (SEQ ID NO: 36) encoding HMGB polypeptide. FIG. 14J is the polypeptide sequence of HMG1L9 (SEQ ID NO: 28) encoding HMGB polypeptide. FIG. 14K is the nucleic acid sequence of LOC122441 (SEQ ID NO: 37) encoding HMGB polypeptide. FIG. 14L is the polypeptide sequence of LOC122441 (SEQ ID NO: 29) encoding HMGB polypeptide. FIG. 14M is the nucleic acid sequence of LOC139603 (SEQ ID NO: 38) encoding HMGB polypeptide. FIG. 14N is the polypeptide sequence of LOC139603 (SEQ ID NO: 30) encoding HMGB polypeptide. FIG. 14O is the nucleic acid sequence of HMG1L8 (SEQ ID NO: 39) encoding HMGB polypeptide. FIG. 14P is the polypeptide sequence of HMG1L8 (SEQ ID NO: 31) encoding HMGB polypeptide.

[Sequence Listing]

Claims (57)

  A high mobility group box protein (HMGM) A box or a variant thereof capable of inhibiting the release of pro-inflammatory cytokines from cells treated with a high mobility group box (HMGB) protein, wherein the HMGB A box comprises A polypeptide selected from the group consisting of: HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box .   A high mobility group box protein (HMGM) A box capable of inhibiting the release of pro-inflammatory cytokines from cells treated with a high mobility group box (HMGB) protein, wherein the HMGB A box comprises HMG1L5 A A polypeptide selected from the group consisting of a box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box.   High mobility group box protein (HMGM) A box biologically active fragment or a polypeptide that is a variant thereof that can inhibit the release of pro-inflammatory cytokines from cells treated with high mobility group box (HMGB) protein. The HMGB A box biologically active fragment is HMG1L5 A box fragment, HMG1L1 A box fragment, HMG1L4 A box fragment, BAC clone RP11-395A23 fragment HMGB A box polypeptide, HMG1L9 A box fragment, LOC122441 A box fragment A polypeptide selected from the group consisting of: a LOC139603 A box fragment, and an HMG1L8 A box fragment.   A polypeptide that is a high mobility group box protein (HMGM) A box biologically active fragment capable of inhibiting the release of pro-inflammatory cytokines from cells treated with a high mobility group box (HMGB) protein, comprising: HMGB A box biologically active fragment is HMG1L5 A box fragment, HMG1L1 A box fragment, HMG1L4 A box fragment, HMGB A box polypeptide fragment of BAC clone RP11-395A23, HMG1L9 A box fragment, LOC122441 A box fragment, LOC139603 A A polypeptide selected from the group consisting of a box fragment and an HMG1L8 A box fragment.   A polypeptide containing a high mobility group box protein (HMGM) A box or a variant thereof capable of inhibiting the release of pro-inflammatory cytokines from cells treated with high mobility group box (HMGB) protein; The HMGB A box is a group consisting of HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box. Selected composition.   A polypeptide containing a high mobility group box protein (HMGM) A box capable of inhibiting the release of pro-inflammatory cytokines from cells treated with the high mobility group box (HMGB) protein, the HMGB A The box is selected from the group consisting of HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box, Composition.   High mobility group box protein (HMGM) A box biologically active fragment capable of inhibiting the release of pro-inflammatory cytokines from cells treated with high mobility group box (HMGB) protein in a pharmaceutically acceptable carrier Or a polypeptide that is a variant thereof, and the HMGB A box biologically active fragment is HMG1L5 A box fragment, HMG1L1 A box fragment, HMG1L4 A box fragment, HMGB A box polypeptide of BAC clone RP11-395A23 A composition selected from the group consisting of a fragment, an HMG1L9 A box fragment, a LOC122441 A box fragment, a LOC139603 A box fragment, and an HMG1L8 A box fragment.   High mobility group box protein (HMGM) A box biologically active fragment capable of inhibiting the release of pro-inflammatory cytokines from cells treated with high mobility group box (HMGB) protein in a pharmaceutically acceptable carrier Wherein the HMGB A box biologically active fragment is HMG1L5 A box fragment, HMG1L1 A box fragment, HMG1L4 A box fragment, HMGB A box polypeptide fragment of BAC clone RP11-395A23, HMG1L9 A composition selected from the group consisting of A box fragment, LOC122441 A box fragment, LOC139603 A box fragment, and HMG1L8 A box fragment.   A purified preparation of an antibody that specifically binds to a high mobility group box protein (HMGB) B box but not specifically to a non-B box epitope of HMGB, wherein the antibody is treated with HMGB A purified preparation, wherein the HMGB B box is selected from the group consisting of an HMG1L5 B box, an HMG1L1 B box, an HMG1L4B box, and an HMGB B box of BAC clone RP11-395A23.   Contains high mobility group box protein (HMGB) B box or variants thereof, but does not contain full-length HMGB and can cause release of pro-inflammatory cytokines from cells, the HMGB B box is HMG1L5 B box, HMG1L1 box A polypeptide selected from the group consisting of HMG1L4 B box, and HMGB B box polypeptide of BAC clone RP11-395A23.   Contains high mobility group box protein (HMGB) B box, but does not contain full-length HMGB and can cause release of pro-inflammatory cytokines from cells, the HMGB B box is HMG1L5 B box, HMG1L1 box, HMG1L4 B A polypeptide selected from the group consisting of a box and an HMGB B box polypeptide of BAC clone RP11-395A23.   A high mobility group box protein (HMGB) B box biological fragment or variant thereof, wherein the HMGB B box biologically active fragment is an HMG1L5 B box fragment, an HMG1L1 box fragment, an HMG1L4 B box fragment, and a BAC clone RP11- A polypeptide selected from the group consisting of 395A23 HMGB B box polypeptide fragments.   High mobility group box protein (HMGB) B box biological fragment, wherein the HMGB B box biologically active fragment is HMG1L5 B box fragment, HMG1L1 box fragment, HMG1L4 B box fragment, and HMGB of BAC clone RP11-395A23 A polypeptide selected from the group consisting of B box polypeptide fragments.   Purified preparation of antibodies that specifically bind to high mobility group box protein (HMGB) B box, but not specifically to non-B box epitopes of HMGB, sufficient to inhibit the inflammatory cytokine cascade The HMGB B box is selected from the group consisting of HMG1L5 B box, HMG1L1 B box, HMG1L4 B box, and HMGB B box polypeptide of BAC clone RP11-395A23. A method of treating a patient condition characterized by activation.   High mobility group box protein capable of inhibiting the release of proinflammatory cytokines from cells treated with high mobility group box (HMGB) protein in an amount sufficient to inhibit the release of proinflammatory cytokines from the cell (HMGB) comprising administering to a patient a polypeptide containing an A box or a variant thereof, wherein the HMGB A box is an HMG1L5 A box, an HMG1L1 A box, an HMG1L4 A box, an HMGB A box polypeptide of the BAC clone RP11-395A23, A method of treating a patient condition characterized by activation of an inflammatory cytokine cascade selected from the group consisting of HMG1L9 A box, LOC122441 B box, LOC139603 A box, and HMG1L8 A box.   High mobility group box protein capable of inhibiting the release of proinflammatory cytokines from cells treated with high mobility group box (HMGB) protein in an amount sufficient to inhibit the release of proinflammatory cytokines from the cell (HMGB) A box comprising administering to the patient a polypeptide that is a biologically active fragment or variant thereof, wherein the HMGB A box is HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, BAC clone RP11-395A23 HMGB A A method of treating a patient condition characterized by activation of an inflammatory cytokine cascade selected from the group consisting of a box polypeptide, an HMG1L9 A box, a LOC122441 A box, a LOC139603 A box, and an HMG1L8 A box.   An effective amount of a polypeptide containing a high mobility group box protein (HMGB) B box or variant thereof sufficient to stimulate the release of proinflammatory cytokines from the cell but not the full-length HMGB polypeptide. Administering to the patient, wherein the HMGB B box results in weight loss in the patient selected from the group consisting of HMG1L5 B box, HMG1L1 B box, HMG1L4 B box, and HMGB B box polypeptide of BAC clone RP11-395A23 Or a method for treating obesity.   Administering to a patient an effective amount of a high mobility group box protein (HMGB) B box biologically active fragment or variant thereof sufficient to stimulate the release of proinflammatory cytokines from the cell Wherein the HMGB B box biologically active fragment is selected from the group consisting of HMG1L5 B box fragment, HMG1L1 B box fragment, HMG1L4 B box fragment, and HMGB B box polypeptide fragment of BAC clone RP11-395A23. A method for effecting weight loss or treating obesity. A compound,
(a) cells that release pro-inflammatory cytokines when exposed to a high mobility group box protein (HMGB) B box or biologically active fragment thereof; and
(b) HMGB B box or biologically active fragment thereof, wherein said HMGB B box is selected from the group consisting of HMG1L5 B box, HMG1L1 B box, HMG1L4 B box, and HMGB B box of BAC clone RP11-395A23;
And then determining whether the compound inhibits the release of pro-inflammatory cytokines from the cell. A method for determining whether a compound inhibits inflammation.   Polypeptides containing high mobility group box (HMGB) A box or fragments or variants thereof and TNF biological activity can inhibit the release of inflammatory cytokines from cells treated with high mobility group box (HMGB) protein A pharmaceutical composition comprising an agent that inhibits, wherein the agent is infliximab, etanercept, adalimumab, CDP870, CDP571, renercept, and thalidomide in a pharmaceutically acceptable carrier.   21. A pharmaceutical composition according to claim 20, wherein the polypeptide is a mammalian HMGB A box.   The pharmaceutical composition according to claim 21, wherein the polypeptide is a mammalian HMGB1 A box.   The pharmaceutical composition according to claim 22, wherein the polypeptide comprises SEQ ID NO: 4.   24. The pharmaceutical composition according to claim 23, wherein the polypeptide comprises SEQ ID NO: 4.   The mammalian HMGB A box consists of HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box The pharmaceutical composition according to claim 21, selected from the group.   Infliximab, etanercept, adalimumab in a pharmaceutically acceptable carrier comprising an antibody that binds to an HMGB polypeptide or a biologically active fragment thereof and an agent that inhibits TNF biological activity, A pharmaceutical composition selected from the group consisting of CDP870, CDP571, Renercept, and thalidomide.   27. The pharmaceutical composition of claim 26, wherein the HMGB polypeptide is a mammalian HMGB polypeptide.   28. The pharmaceutical composition according to claim 27, wherein the HMGB polypeptide is an HMGB1 polypeptide.   29. The pharmaceutical composition according to claim 28, wherein the HMGB1 polypeptide comprises SEQ ID NO: 1.   30. The pharmaceutical composition according to claim 29, wherein said HMGB1 polypeptide comprises SEQ ID NO: 1.   27. The pharmaceutical composition according to claim 26, wherein the biologically active HMGB fragment is an HMGB B box or a biologically active fragment thereof.   32. The pharmaceutical composition according to claim 31, wherein the HMGB B box consists of SEQ ID NO: 5.   32. The pharmaceutical composition of claim 31, wherein said HMGB B box biological fragment consists of SEQ ID NO: 23.   32. The pharmaceutical composition according to claim 31, wherein the HMGB B box is selected from the group consisting of HMG1L5 B box, HMG1L1 B box, HMG1L4 B box, and HMGB B box polypeptide of BAC clone RP11-395A23.   27. The pharmaceutical composition according to claim 26, wherein the antibody is a monoclonal antibody.   27. The pharmaceutical composition according to claim 26, wherein the antibody is a polyclonal antibody.   Polypeptides containing high mobility group box (HMGB) A box or fragments or variants thereof and TNF biological that can inhibit the release of proinflammatory cytokines from cells treated with high mobility group box (HMGB) protein Activating an inflammatory cytokine cascade comprising administering to a patient a composition comprising an agent that inhibits activity, wherein the agent is selected from the group consisting of infliximab, etanercept, adalimumab, CDP870, CDP571, renercept, and thalidomide A method of treating a condition in a patient characterized by:   38. The method of claim 37, wherein the composition further comprises a pharmaceutically acceptable carrier.   38. The method of claim 37, wherein the polypeptide is a mammalian HMGB A box.   40. The method of claim 39, wherein the polypeptide is a mammalian HMGB1 A box.   41. The method of claim 40, wherein the polypeptide comprises SEQ ID NO: 4.   42. The method of claim 41, wherein the polypeptide consists of SEQ ID NO: 4.   The mammalian HMGB A box consists of HMG1L5 A box, HMG1L1 A box, HMG1L4 A box, HMGB A box polypeptide of BAC clone RP11-395A23, HMG1L9 A box, LOC122441 A box, LOC139603 A box, and HMG1L8 A box 40. The method of claim 39, selected from the group.   Said condition is sepsis, graft rejection, rheumatoid arthritis, asthma, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, organ ischemia, reperfusion ischemia 38. The method of claim 37, selected from the group consisting of: Behcet's disease, graft versus host disease, Crohn's disease, ulcerative colitis, multiple sclerosis, and cachexia.   Administering to a patient a composition comprising an antibody that binds to an HMGB polypeptide or a biologically active fragment thereof and an agent that inhibits TNF biological activity, wherein the agent is infliximab, etanercept, adalimumab, CDP870, CDP571 A method for the treatment of a condition in a patient characterized by activation of an inflammatory cytokine cascade selected from the group consisting of, Renercept, and thalidomide.   46. The method of claim 45, wherein the composition further comprises a pharmaceutically acceptable carrier.   46. The method of claim 45, wherein the (HMGB) polypeptide is a mammalian HMGB polypeptide.   48. The method of claim 47, wherein the HMGB polypeptide is an HMGB1 polypeptide.   49. The method of claim 48, wherein the HMGB1 polypeptide contains SEQ ID NO: 1.   52. The method of claim 49, wherein the HMGB1 polypeptide consists of SEQ ID NO: 1.   46. The method of claim 45, wherein the biologically active HMGB fragment is an HMGB B box or a biologically active fragment thereof.   52. The method of claim 51, wherein the HMGB B box is selected from HMG1L5 B box, HMG1L1 B box, HMG1L4B box, RP11-395A23 HMGB B box polypeptide.   52. The method of claim 51, wherein the HMGB1 B box consists of SEQ ID NO: 5.   52. The method of claim 51, wherein the HMGB1 B box biologically active fragment consists of SEQ ID NO: 23.   46. The method of claim 45, wherein the antibody is a monoclonal antibody.   46. The method of claim 45, wherein the antibody is a polyclonal antibody. Said condition is sepsis, graft rejection, rheumatoid arthritis, asthma, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, organ ischemia, reperfusion ischemia 46. The method of claim 45, selected from the group consisting of: Behcet's disease, graft versus host disease, Crohn's disease, ulcerative colitis, multiple sclerosis, and cachexia.