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WO2005025604A2 - Utilisation de hmgb et fragments de hmgb pour faire diminuer la reponse immunitaire specifique - Google Patents

Utilisation de hmgb et fragments de hmgb pour faire diminuer la reponse immunitaire specifique Download PDF

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Publication number
WO2005025604A2
WO2005025604A2 PCT/US2004/029540 US2004029540W WO2005025604A2 WO 2005025604 A2 WO2005025604 A2 WO 2005025604A2 US 2004029540 W US2004029540 W US 2004029540W WO 2005025604 A2 WO2005025604 A2 WO 2005025604A2
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hmgb
box
polypeptide
seq
individual
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PCT/US2004/029540
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WO2005025604A3 (fr
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Howland Shaw Warren, Jr.
Kevin J. Tracey
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The General Hospital Corporation
North Shore-Long Island Jewish Research Institute
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Publication of WO2005025604A2 publication Critical patent/WO2005025604A2/fr
Publication of WO2005025604A3 publication Critical patent/WO2005025604A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the body's immune system attacks substances recognized by the body as foreign (antigens). This is highly problematic when a therapeutically administered antigen is perceived by a patient as foreign, resulting in a detrimental immune response.
  • the detrimental response to the administered antigen can be mediated by components of the immune system, either cells or antibodies (humoral response). Immune pathologies can be induced by therapeutic administration of a wide variety of antigens to an individual. For example, administration of a non-human antibody can lead to a detrimental response. For instance, a human anti-mouse antibody response (HAMA) can result from administration of a mouse IgG molecule.
  • HAMA human anti-mouse antibody response
  • an immune pathology can be caused by therapeutic administration of any material to an individual which is recognized by the individual's immune system as non-self material. This can lead to serious problems, including allograft rejection, or a detrimental response to a blood transfusion.
  • Therapeutic administration of an adjuvant can also lead to a detrimental immune response.
  • An infection can also induce a specific immune response which is potentially harmful to the host by inducing an autoimmune response. It would be useful to treat a detrimental immune response of an individual to a foreign antigen.
  • a high mobility group box protein (HMGB) molecule can be used therapeutically to decrease the specific immune response to an antigen.
  • the invention comprises administration of HMGB, or isolated portions of it such as the A box, to decrease the specific immune response of an individual, e.g., a host (animal or human), to a foreign antigen.
  • Specific immune responses include a humoral (antibody) response and a cellular response.
  • the invention includes use of the HMGB protein in a prophylactic sense (administered before or with the antigen), or in a treatment sense (after the immune response has already started).
  • the present invention is directed to treating (e.g., inhibiting, alleviating or preventing) an immune pathology in an individual, comprising administering to the individual an effective amount of a high mobility group box protein ( ⁇ MGB) polypeptide comprising a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment of a vertebrate HMGB A box or non-naturally occurring HMGB A box.
  • ⁇ MGB high mobility group box protein
  • the HMGB polypeptide can be any HMGB polypeptide described herein which comprises a vertebrate or non-naturally occurring HMGB A box or fragment.
  • the HMGB polypeptide is an HMGBl polypeptide.
  • the HMGB polypeptide can be a full length peptide. It can be a wild type polypeptide or a non- naturally occurring peptide (e.g., a functional equivalent), including a fragment, variant or mutation of the wild type polypeptide.
  • the HMGB polypeptide is a wild type HMGB polypeptide (e.g., HMGBl polypeptide).
  • the HMGB polypeptide comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 6.
  • the HMGB A box can be a mammalian HMGB A box, such as a human HMGB A box.
  • the HMGB A box is an HMGBl A box, such as a mammalian HMGB 1 A box, for example, a human HMGB 1 A box.
  • the HMGBl A box comprises or consists of the sequence of SEQ LD NO:4 or SEQ ID NO:7.
  • the HMGB polypeptide comprises or consists of the A box and the amino terminus of the HMGB polypeptide (e.g., the HMGBl polypeptide).
  • the HMGB polypeptide comprises a vertebrate HMGB B box.
  • the HMGB B box is a mammalian HMGB B box, for example, a human HMGB B box.
  • the HMGB B box can be an HMGB 1 B box, such as a mammalian HMGBl B box, e.g., a human HMGBl B box.
  • the HMGBl B box comprises or consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:8.
  • the HMGB polypeptide comprises a vertebrate HMGB
  • HMGB polypeptide is truncated at the amino terminus, the carboxyl terminus, or both termini, the truncation being relative to the full length HMGB polypeptide.
  • the HMGB polypeptide comprises a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment thereof; and a vertebrate HMGB B box or a non-naturally occurring HMGB B box, wherein the HMGB polypeptide is truncated at the carboxyl terminus, the truncation being relative to the full length HMGB polypeptide (e.g., a "nAB" fragment).
  • the HMGB polypeptide comprises or consists of SEQ ID NO: 57.
  • the "immune pathology" can be any detrimental response to an antigen by components of the immune system, either cells or antibodies, but the term excludes autoimmune disorders.
  • the immune pathology is induced by therapeutic administration of a non-human antigen to the individual.
  • a non-human antigen refers to an agent (e.g., a molecule) capable of stimulating an immune response.
  • Therapeutic administration includes administration for purposes of, or relating to, treatment or prophylaxis (prevention) of a disease, disorder, pathology or detrimental condition.
  • the methods can be used to decrease the specific immune response to a protein that is administered for therapeutic purposes.
  • the non-human antigen is an antibody, an immunoglobulin molecule or an immunogenic fragment thereof.
  • antibody i. e.
  • an immunoglobulin refers to a protein that reacts with a specific antigen.
  • the antibody can be of any type or class of immunoglobulin molecule.
  • the term "antibody” includes mutations and variants, including chimeric, humanized, optimized and engineered antibodies, and fusion proteins comprising an immunoglobulin or a fragment thereof.
  • the administration of the HMGB polypeptide could decrease the human anti-mouse antibody response (HAMA) resulting from administration of a non-human (mouse) antibody (e.g., mouse IgG).
  • the immune pathology treated is induced by transplantation (for example, transplantation of an organ) into the individual.
  • Transplantation refers to the transfer (i.e., administration) of protein (e.g., cells, tissues, or organs) from one organism (the donor) to another (the recipient) or from one part of the body to another (for example, skin grafts).
  • protein e.g., cells, tissues, or organs
  • organ transplantation a foreign protein transplanted into humans can induce a specific immune response to the protein.
  • HMGB e.g., HMGBl
  • immune pathology is induced by therapeutic administration of a material to the individual which is recognized by the individual's immune system as non-self material.
  • the material administered to the individual can be a cell (e.g., a transfusion), a tissue or any other substance in which there is the risk of an induced specific immune response to the administered foreign material, where "foreign” means any material recognized as non-self.
  • the material administered is bone marrow cells.
  • the immune pathology is induced by therapeutic administration of an adjuvant to the individual.
  • adjuvant refers to a substance which increases the immune response.
  • the immune pathology is induced by infection from a microorganism (e.g., a bacteria, virus or fungus). In some embodiments, the infection induces a specific immune response.
  • autoimmune disorders e.g. , a disorder wherein the individual's immune system malfunctions, misinterprets a material in the I individual's body as foreign, and attacks it, resulting in an autoimmune reaction.
  • the autoimmune disorder is selected from the group consisting of allergy, ulcerative colitis, Crohn's disease, asthma, rheumatoid arthritis, psoriasis and systemic lupus erythematosus (SLE).
  • Other autoimmune disorders include myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, scleroderma, Sjogren's syndrome, and pernicious anemia.
  • the immune pathology is induced by an allergic response.
  • the invention relates to a method of protecting a subject against an immune pathology, comprising administering to the individual an effective amount of a high mobility group box protein (HMGB) polypeptide comprising a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment of said vertebrate HMGB A box or non-naturally HMGB A box.
  • HMGB high mobility group box protein
  • the invention in another embodiment, relates to a method of inhibiting an immune pathology in an individual, comprising administering to the individual an effective amount of an HMGB polypeptide comprising a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment of said vertebrate HMGB A box or non-naturally HMGB A box.
  • the invention in another embodiment, relates to a method of decreasing an immune response to an administered non-human antigen, comprising administering to an individual an effective amount of a HMGB polypeptide comprising a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment of said vertebrate HMGB A box or non-naturally HMGB A box.
  • the invention in another embodiment, relates to a method of treating an immune pathology in an individual, comprising administering to the individual an effective amount of a HMGB polypeptide comprising a vertebrate HMGB A box, a non-naturally occurring HMGB A box, or an immunosuppressive fragment thereof; and a vertebrate HMGB B box or a non-naturally occurring HMGB B box.
  • the HMGB polypeptide is a HMGBl peptide.
  • the HMGB polypeptide is truncated at the carboxyl terminus.
  • FIG. 1A is the amino acid sequence of a human HMGl polypeptide (SEQ ID NO:l).
  • FIG. IB is the amino acid sequence of rat and mouse HMGl (SEQ ID NO:2).
  • FIG. IC is the amino acid sequence of human HMG2 (SEQ ID NO:3).
  • FIG. ID is the amino acid sequence of a human, mouse, and rat HMGl A box polypeptide (SEQ ID NO:4).
  • FIG. IE is the amino acid sequence of a human, mouse, and rat HMGl B box polypeptide (SEQ ID NO:5).
  • IF is the nucleic acid sequence (SEQ ID NO:46) of a forward primer for human HMGl (also known as “HOLO HMG” or “Ml”).
  • FIG. 1G is the nucleic acid sequence (SEQ ID NO:47) of a reverse primer for human HMGl (also known as “HOLO HMG” or “Ml”).
  • FIG. 1H is the nucleic acid sequence (SEQ ID NO:48) of a forward primer for the carboxyl terminus mutant of human HMGl (also known as "nAB” or “M2”).
  • FIG. II is the nucleic acid sequence (SEQ ID NO:49) of a reverse primer for the carboxyl terminus mutant of human HMGl (also known as "nAB” or "M2").
  • FIG. 1 J is the nucleic acid sequence (SEQ ID NO:50) of a forward primer for the A box plus B box mutant of human HMGl (also known as “AB” or “M4").
  • FIG. IK is the nucleic acid sequence (SEQ TD NO:51) of a reverse primer for the A box plus B box mutant of human HMGl (also known as “AB” or “M4").
  • FIG. IL is the nucleic acid sequence (SEQ ID NO: 52) of a forward primer for a B box mutant of human HMGl (also known as “B” or “M5").
  • FIG. 1M is the nucleic acid sequence (SEQ ID NO:53) of a reverse primer for a B box mutant of human HMGl (also known as "B” or "M5").
  • FIG. IN is the nucleic acid sequence (SEQ ID NO: 54) of a forward primer for the amino terminus plus A box mutant of human HMGl (also known as “A” or “M6”).
  • FIG. 10 is the nucleic acid sequence (SEQ ID NO: 55) of a reverse primer for the amino terminus plus A box mutant of human HMGl (also known as "A” or “M6”).
  • FIG. 2A is the nucleic acid sequence of HMGILIO (SEQ ID NO: 9) encoding an HMGB polypeptide.
  • FIG. 2B is the polypeptide sequence of HMGILIO (SEQ ID NO: 10) encoding an HMGB polypeptide.
  • FIG. 2C is the nucleic acid sequence of HMG1L1 (SEQ ID NO: 11) encoding an HMGB polypeptide.
  • FIG. 2D is the polypeptide sequence of HMG1L1 (SEQ ID NO: 12) encoding an HMGB polypeptide.
  • FIG. 2E is the nucleic acid sequence of HMG1L4 (SEQ ID NO: 13) encoding an HMGB polypeptide.
  • FIG. 2F is the polypeptide sequence of HMG1L4 (SEQ ID NO: 14) encoding an HMGB polypeptide.
  • FIG. 2G is the nucleic acid sequence of the HMG polypeptide sequence of the BAC clone RPl 1-395A23 (SEQ ID NO: 15).
  • FIG. 2H is the polypeptide sequence of the HMG polypeptide sequence of the BAC clone RPl 1-395A23 (SEQ ID NO: 16) encoding an HMGB polypeptide.
  • FIG. 21 is the nucleic acid sequence of HMG1L9 (SEQ ID NO: 17) encoding an HMGB polypeptide.
  • FIG. 2J is the polypeptide sequence of HMG1L9 (SEQ ID NO: 18) encoding an HMGB polypeptide.
  • FIG. 2K is the nucleic acid sequence of LOC122441 (SEQ ID NO: 19) encoding an HMGB polypeptide.
  • FIG. 2L is the polypeptide sequence of LOC122441 (SEQ ID NO: 20) encoding an HMGB polypeptide.
  • FIG. 2M is the nucleic acid sequence of LOC139603 (SEQ ID NO: 21) encoding an HMGB polypeptide.
  • FIG. 2N is the polypeptide sequence of LOC139603 (SEQ ID NO: 22) encoding an HMGB polypeptide.
  • FIG. 2O is the nucleic acid sequence of HMG1L8 (SEQ ID NO: 23) encoding an HMGB polypeptide.
  • FIG. 2P is the polypeptide sequence of HMGl L8 (SEQ ID NO: 24) encoding an HMGB polypeptide.
  • FIG. 3 is a sequence alignment of HMGl polypeptide sequence from rat (SEQ ID NO:2), mouse (SEQ ID NO:2), and human (SEQ ID NO:6).
  • FIG. 4 is a sequence alignment of human HMGB homologous proteins
  • FIG. 5 is a histogram of the effects of HMGB-1, including full length HMG and HMGB fragments, alone or in combination with peptidoglycan-associated lipoprotein (PAL) on C3H/HEJ mouse splenocytes. These effects are compared with the effects of media, PAL alone or lipopolysaccharide (LPS) (E. coli 04) (expressed as counts per minute).
  • FIG. 6 depicts times of bleeds and immunization protocol for HMGBl antibody studies described in Example 3.
  • FIG. 8 depict the effect of the nAB (A box plus B box plus amino terminus) fragment of HMGB on anti-ovalbumin antibody response in C3H HEJ mice, measured as fold rise in titer to ovalbumin.
  • the data for the 10-13 mice per group in Figure 8 includes the data for the 3 mice per group in Figure 7.
  • FIG. 9 is a histogram of the effects of HMGl nAB on antibody response in 4 mice injected intramuscularly with tetanus toxoid (TT) (left bars) and peptidoglycan- associated lipoprotein (PAL) (right bars) (in the same thigh) at 4 weeks and six weeks, measured as fold rise in titer.
  • TT tetanus toxoid
  • PAL peptidoglycan- associated lipoprotein
  • the present invention is based on the discovery that HMGBl, or portions of it, when administered together with an antigen, diminish the antibody response to the administered antigen. Therefore, HMGB, or portions of the molecule, can be used therapeutically to decrease the specific immune response to an antigen.
  • the invention comprises administration of HMGB, or isolated portions of it such as the A box, with the goal of decreasing the specific immune response of an individual, e.g., a host (animal or human) to any foreign antigen.
  • Specific immune response includes a humoral (antibody) response or a cellular response.
  • the invention includes use of the protein in a prophylactic sense (administered before or with the antigen), or in a treatment sense (after the immune response has already started).
  • High mobility group- 1 HMGl
  • HMG-1 and HMGBl High mobility group- 1
  • HMG high mobility group
  • HMGBl The HMGBl molecule has three domains: two DNA binding motifs termed HMGB A and HMGB B boxes, and an acidic carboxyl terminus.
  • the two HMGB boxes are highly conserved 80 amino acid, L-shaped domains.
  • HMGB boxes are also expressed in other transcription factors, including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.
  • HMGBl is a potent activator of monocytes. It has also been implicated as a ligand for RAGE, a multi-ligand receptor of the immunoglobulin superfamily.
  • RAGE is expressed on endothelial cells, smooth muscle cells, monocytes, and nerves, and ligand interaction transduces signals through MAP kinase, P21 ras, and NF-KB.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell culture, molecular biology, microbiology, cell biology, and immunology, which are well within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Sambrook, J. & Russell, D.W. Molecular Cloning: a Laboratory Manual (3rd ed.) NY: Cold Spring Harbor Laboratory Press (2001); Ausubel, F.M. et al.
  • HMGB POLYPEPTIDES An "HMGB polypeptide” refers to a full length HMGB protein or any fragment thereof.
  • a "HMGB protein” also known as HMG protein
  • HMG protein is a protein or a polypeptide which has at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a sequence described herein, such as a sequence selected from SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 6
  • sequence identity can be determined, for example, using the BLAST program and parameters described herein.
  • polypeptide refers to a polymer of amino acids, and not to a specific length.
  • peptides, oligopeptides and proteins are included within the definition of a polypeptide.
  • the polypeptide is preferably a vertebrate polypeptide, for example, a mammalian polypeptide, such as a human polypeptide.
  • the HMGB polypeptide or protein is a vertebrate such as a mammalian (e.g., human) HMGB polypeptide.
  • the HMGB polypeptide or protein is HMGB 1.
  • Examples of an HMGB polypeptide include a polypeptide comprising or consisting of the sequence of SEQ LO NO:l, SEQ TD NO:2, SEQ TD NO:3, SEQ ID NO:6 or SEQ ID NO:57.
  • the HMGB polypeptide can contain a B box DNA binding domain and/or an A box DNA binding domain, and/or an acidic carboxyl terminus as described herein. See, for example, Bianchi et al, "The DNA binding site of HMGl protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins," The EMBO Journal, 11(3): 1055-1063 (1992), the entire contents of which are incorporated herein by reference in their entirety. Other examples of HMGB polypeptides and proteins are described in
  • HMGB polypeptides include, but are not limited to, mammalian HMGl ((HMGBl) as described, for example, in GenBank Accession Number U51677), HMG2 ((HMGB2)as described, for example, in GenBank Accession Number M83665), HMG-2A ((HMGB3, HMG-4) as described, for example, in GenBank Accession Number NM_005342 and NP_005333), HMG14 (as described, for example, in GenBank Accession Number P05114), HMGl 7 (as described, for example, in GenBank Accession Number X13546), HMGl (as described, for example, in GenBank Accession Number L17131), and HMGY (as described, for example, in GenBank Accession Number M23618); nonmammalian HMG Tl (as described, for example, in GenBank Accession Number X02666) and HMG T2 (as described, for example, in GenBank Accession Number L32859) (
  • HMGB polypeptides and proteins also include, but are not limited to, polypeptides encoded by HMGB nucleic acid sequences GenBank Accession Numbers NG_00897 (HMGILIO) (and in particular by nucleotides 658- 1305 of NG_00897, as shown in FIGs. 2A and 2B); AF076674 (HMGILI) (and in particular by nucleotides 1-633 of AF076674, as shown in FIGs. 2C and 2D); AF076676 (HMG1L4 ) (and in particular by nucleotides 1-564 of AF076676, as shown in FIGs.
  • HMGILIO GenBank Accession Numbers NG_00897
  • AF076674 HMGILI
  • HMG1L4 AF076676
  • AC010149 HMG sequence from BAC clone RPl 1- 395A23 (and in particular by nucleotides 75503-76117 of AC010149, as shown in FIGs. 2G and 2H); AF 165168 (HMG1L9) (and in particular by nucleotides 729-968 of AF165168, as shown in FIGs. 21 and 2J); XM_063129 (LOC122441) (and in particular by nucleotides 319-558 of XM_063129, as shown in FIGs.
  • HMGB-like sequences in such polypeptides can be determined and isolated using methods described herein, for example, by performing sequence comparisons to HMGB polypeptides described herein and testing for biological activity using a method described herein or other method known in the art.
  • the HMGB polypeptide is a substantially pure, or substantially pure and isolated polypeptide that has been separated from components that naturally accompany it.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is separated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a "fusion protein") and still be “isolated” or “purified.” It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful.
  • the polypeptide may be in an unpurified form, for example, in a cell, cell milieu, or cell extract.
  • HMGB polypeptides can be purified from cells that naturally " express HMGB, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell and the polypeptide is expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • HMGB FUNCTIONAL EQUIVALENTS Functional equivalents of HMGB (proteins or polypeptides that have one or more of the biological activities of a wild type HMGB polypeptide), including non-naturally occurring HMGB proteins and polypeptides, can also be used in the methods of the present invention.
  • Biologically active fragments, sequence variants, post-translational modifications, and chimeric or fusion proteins comprising the protein, biologically active fragment, or variant are examples of functional equivalents of a protein.
  • Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants.
  • variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to the protein of interest, for example, an HMGB protein as described herein.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • variant polypeptides can be fully functional or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Functional variants can also contain substitution of similar amino acids (e.g., conservative amino acid substitutions) that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al , Science, 244: 1081 - 1085 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol, 224:899-904 (1992); and de Vos et al, Science, 255:306-312 (1992).
  • FRAGMENTS Fragments of HMGB proteins which comprise the A box can also be used in the present invention.
  • a fragment comprises at least 6 contiguous amino acids from an HMGB polypeptide.
  • Useful fragments include those that retain one or more of the biological activities of the polypeptide.
  • Examples of HMGB biologically active fragments include the A box, the B box, immunosuppressive active fragments of the A box and B box, and fragments comprising the A box and the B box (including fragments in which the polypeptide is truncated at the carboxyl terminus and/or the amino terminus), and fragments comprising the amino terminus and the A box and/or the B box.
  • Biologically active e.g., immunosuppressive, fragments (peptides which are, for example, 6, 9, 12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) (including C-terminal truncated fragments and/or N-terminal truncated fragments) can comprise one or more domains, segments, or motifs that have been identified by analysis of the polypeptide sequence using well-known methods, e.g., signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, or post-translation modification sites.
  • domains include the A box and/or the B box as described herein.
  • the fragment does not include the amino terminus (e.g., the first eight amino acids of the wild type HMGBl sequence). Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and/or pro-polypeptide regions fused to the an ⁇ ino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment. For example, a fragment can decrease the specific immune response of a host (animal or human) to any foreign antigen.
  • the specific immune response includes either a humoral (antibody) response or a cellular response.
  • Biologically active fragments can be generated using standard molecular biology techniques and assaying the function of the fragment, (for instance, by determining if the fragment, when administered to a host, inhibits a specific immune response), using, for example, methods described herein.
  • Immunosuppressive fragments can be used in the methods described herein in which full length polypeptides are used, for example, inhibiting an immune response by a cell, or treating a patient having a condition characterized by an immune pathology.
  • the invention also provides uses and methods for chimeric or fusion polypeptides containing an HMGB polypeptide or a functional equivalent of HMGB.
  • These chimeric proteins comprise an HMGB polypeptide or functional equivalent thereof operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide. "Operatively linked" indicates that the polypeptide and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In one embodiment the fusion polypeptide does not affect function of the HMGB polypeptide per se.
  • the fusion polypeptide can be a GST-fusion polypeptide in which the HMGB polypeptide sequences are fused to the C-terminus of the GST sequences.
  • Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example, ⁇ -galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, FLAG-tagged fusions, GFP fusions, and Ig fusions.
  • Such fusion polypeptides can facilitate the purification of a recombinant polypeptide.
  • host cells e.g.
  • the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
  • HMGB, HMGB A box and HMGB B box functional equivalents either naturally occurring or non-naturally occurring, include polypeptides that have sequence identity to the HMGB polypeptides, HMGB A boxes and HMGB B boxes described herein.
  • two polypeptides are substantially homologous or identical when the amino acid sequences are at least about 60%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous or identical.
  • the length of the HMGB polypeptide, HMGB A box polypeptide, or HMGB B box polypeptide aligned for comparison purposes is at least 30%, preferably, at least 40%, more preferably, at least 60%, and even more preferably, at least 70%, 80%, 90%, or 100% of the length of the reference sequence, for example, those sequence provided in FIGS. 1A-1E, and SEQ ID NOS: 6, 7, and 8.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using 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) as described in Schaffer et al, Nucleic Acids Res., 29:2994-3005 (2001).
  • BLASTN the default parameters of the respective programs
  • the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
  • NR non-redundant
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California) 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.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California), using a gap weight of 50 and a length weight of 3.
  • HMGB functional equivalents can be generated using standard molecular biology techniques and the function can be assayed using, for example, methods described herein, such as determining if the functional equivalent, when administered to a cell (e.g., a macrophage) inhibits the immune response from the cell, compared to an untreated control cell.
  • the HMGB functional equivalent has at least 50%, 60%, 70%, 80%, or 90% of the biological activity of the HMGBl polypeptide of SEQ ID NO: 1.
  • HMGB A BOX The methods of the present invention also feature HMGB A boxes.
  • an "HMGB A box”, also referred to herein as an “A box” is a protein or polypeptide that has at least 50%, 60%, 70%, 75%, 80%», 85%), 90%», or 95% sequence identity to an HMGB A box as described herein.
  • the HMGB A box has no more than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the biological activity of full length HMGB.
  • the HMGB A box amino acid consists of the sequence of SEQ ID NO: 4 or SEQ ID NO:7 (PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSE RWKTMS AKEKGKFEDMAKADKARYEREMKTYIPPKGET) or the amino acid sequence in the corresponding region of an HMGB protein in a mammal.
  • An HMGB A box is also a recombinantly produced polypeptide having the same amino acid sequence as the A box sequences described above.
  • the HMGBl A box comprises or consists of the sequence of SEQ ID NO: 4 or SEQ JJD NO: 7.
  • the A box has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ LD NOs: 4 or 7.
  • An HMGB A box often has no more than about 85 amino acids and no fewer than about 4 amino acids.
  • polypeptides having A box sequences within them include, but are not limited to GenBank Accession Numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP_002119, CAA31110, S02826, U00431, X67668, NP_005333, NM_016957, and J04197; HMGB polypeptides described herein; mammalian HMGl ((HMGBl) as described, for example, in GenBank Accession Number U51677), HMG2 ((HMGB2)as described, for example, in GenBank Accession Number M83665), HMG-2A ((HMGB3, HMG- 4) as described, for example, in GenBank Accession Numbers NM_005342 and NP_005333), HMG14 (as described, for example, in GenBank Accession Number P05114), HMGl 7 (as described, for example, in GenBank Accession Number X13546), HMGMG
  • polypeptides having A box sequences within them include, but are not limited to, polypeptides encoded by GenBank Accession Numbers NG_00897 (HMGILIO) (and, in particular, by nucleotides 658-1305 of NG_00897, as shown in FIGs. 2A and 2B); AF076674 (HMGILI) (and in particular by nucleotides 1-633 of AF076674, as shown in FIGs. 2C and 2D; AF076676 (HMG1L4 ) (and, in particular, by nucleotides 1-564 of AF076676, as shown in
  • FIGs. 2E and 2F AC010149 (HMG sequence from BAC clone RPl 1-395A23) (and in particular by nucleotides 75503-76117 of AC010149), as shown in FIGs. 2G and 2H); AF165168 (HMG1L9) (and in particular by nucleotides 729-968 of AF165168, as shown in FIGs. 21 and 2J); XM_063129 (LOC122441) (and in particular by nucleotides 319-558 of XM_063129, as shown in FIGs.
  • a box sequences in such polypeptides can be determined and isolated using methods described herein, for example, by sequence comparisons to A boxes described herein and testing for biological activity using methods described herein or other methods known in the art.
  • HMGB A box polypeptide sequences include the following sequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (Human HMGBl; SEQ ID NO: 25); DSSVNFAEF SKKCSERWKT MS AKEKSKFE DMAKSDKARY DREMKNYVPP KGDK; (Human HMGB2; SEQ ID NO: 26); PEVPVNFAEF SKKCSERWKT VSGKEKSKFD EMAKADKVRY DREMKDYGPA KGGK (Human HMGB3; SEQ ID NO: 27); PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (HMGILIO; SEQ ID NO: 28); SDASVNFSEF SNKCSERWKT MSAKEKGKFE DMAKADKTHY ERQMKTYIPP KGET (HMG1L1; SEQ ID NO: 29); PDASV
  • An HMGB A box (or A box) can also be a substantially pure, or substantially pure and isolated polypeptide that has been separated from components that naturally accompany it, and which consists of an amino acid sequence that is less than a full length HMGB polypeptide and which suppresses an immune response.
  • Non-naturally occurring HMGB A boxes can also be used in the methods of the present invention.
  • a functional equivalent of an HMGB A box inhibits an immune response from a cell contact with an antigen.
  • HMGB A box functional equivalents include, for example, immunosuppressive fragments, post-translational modifications, variants, or fusion proteins comprising A boxes, as defined herein.
  • an "A box fragment that has A box biological activity” or an "A box immunosuppresssive fragment” is meant a fragment of an HMGB A box that has the activity of an HMGB A box, as described herein.
  • a box functional equivalents can be generated using standard molecular biology techniques and their function can be assayed using known methods, for example, by determining if the fragment, when administered to an individual or a cell (e.g., a macrophage) decreases or inhibits an immune response by the cell.
  • the A box functional equivalent has at least 50%, 60%, 70%, 80%, 90% or 95% of the biological activity of the HMGBl polypeptide of SEQ ID NO: 4, SEQ ID NO:7 OR SEQ ID NO:36 determined using the BLAST program and parameters described herein, and has one of more of the biological activities of an HMGB A box.
  • the HMGB A box polypeptide is a substantially pure, or substantially pure and isolated polypeptide that has been separated from components that naturally accompany it.
  • the polypeptide may be in an unpurified form, for example, in a cell, cell milieu, or cell extract.
  • the critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components.
  • HMGB B BOX The methods of the present invention also feature HMGB B boxes.
  • an "HMGB B box” also referred to herein as a "B box” (and also known as an HMG B box) is a polypeptide that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOS: 5 or 8 (as determined using the BLAST program and parameters described herein) and lacks an A box. In one embodiment, it inhibits the immune response of a cell.
  • the HMGB B box has at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the biological activity of full length HMGB.
  • the HMGB B box is a polypeptide that is about 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, or 20% the length of a full length HMGBl polypeptide.
  • the HMGB B box comprises or consists of the sequence of SEQ ID NO: 5, SEQ ID NO: 8 (FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAAD DKQPYEKKAAKLKEKYEKDIAAY) or SEQ ID NO: 43, or the amino acid sequence in the corresponding region of an HMGB protein in a mammal.
  • the polypeptide comprises or consists of a specific polypeptide sequence of the HMGBl B box, comprising amino acids 1-20 of SEQ ID NO:8 (SEQ ID NO:45), or comprising amino acids 1-20 of SEQ ID NO:5 (SEQ ID NO:44), or consisting of amino acids 1-20 of SEQ ID NO:8 (SEQ ID NO:45), or consisting of amino acids 1 -20 of SEQ ID NO:5 (SEQ ID NO:44).
  • An HMGB B box polypeptide can also be a recombinantly produced polypeptide having the same amino acid sequence as an HMGB B box polypeptide described above, and which is a portion of a full length HMGB polypeptide and comprises or consists of the B box domain of HMGB.
  • An HMGB B box (or B box) also can be a substantially pure, or substantially pure and isolated polypeptide that has been separated from components that naturally accompany it, and which consists of an amino acid sequence that is less than a full length HMGB polypeptide and which suppresses an immune response.
  • the HMGB B box is a mammalian HMGB B box, for example, a human HMGBl B box.
  • An HMGB B box often has no more than about 85 amino acids and no fewer than about 4 amino acids.
  • polypeptides having B box sequences within them include, but are not limited to, GenBank Accession Numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP_002119, CAA31110, S02826, U00431, X67668, NP_005333, NM_016957, and J04197; HMG polypeptides described herein; mammalian HMGl ((HMGBl) as described, for example, in GenBank Accession Number U51677), HMG2
  • HMG2 as described, for example, in GenBank Accession Number M83665)
  • HMG-2A ((HMGB3, HMG-4) as described, for example, in GenBank Accession Numbers NM_005342 and NP_005333)
  • HMG14 as described, for example, in GenBank Accession Number P05114
  • HMG17 as described, for example, in GenBank Accession Number XI 3546
  • HMGl as described, for example, in
  • GenBank Accession Number LI 7131 GenBank Accession Number LI 7131
  • HMGY HMGY
  • nonmammalian HMG Tl HMG Accession Number X02666
  • HMG T2 HMG T2
  • HMG-X HMG-X
  • HMG D HMG D
  • HMG Z HMG Z
  • NHPIO protein HMG protein homolog NHP 1
  • non-histone chromosomal protein as described, for example, in GenBank Accession Number O00479)
  • HMG 1/ 2 like protein as described, for example, in GenBank Accession Number
  • polypeptides having B box sequences within them include, but are not limited polypeptides encoded by GenBank Accession Numbers NG_00897 (HMGILIO) (and in particular by nucleotides 658-1305 of NG_00897, as shown in FIGs. 2A and 2B); AF076674 (HMGILI) (and in particular by nucleotides 1-633 of AF076674, as shown in FIGs. 2C and 2D; AF076676 (HMG1L4 ) (and in particular by nucleotides 1-564 of AF076676, as shown in FIGs.
  • HMGB B box polypeptide sequences include the following sequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (human HMGBl; SEQ ID NO: 37); KKDPNAPKRP PSAFFLFCSE HRPKIKSEHP GLSIGDTAKK
  • LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY (human HMGB2; SEQ ID NO: 38); FKDPNAPKRL PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMGILIO; SEQ ID NO: 39); FKDPNAPKRP PSAFFLFCSE YHPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPGEKKA AKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 40); FKDSNAPKRP PSAFLLFCSE YCPKTKGEHP GLPISDVAKK
  • LVEMWNNTFA DDKQLCEKKA AKLKEKYKKD TATY HMG1L4; SEQ ID NO: 41
  • FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVVKK LAGMWNNTAA ADKQFYEKKA AKLKEKYKKD IAAY HMG sequence from BAC clone RPl 1-359A23; SEQ ID NO: 42
  • HMGBl box human HMGBl box; SEQ ID NO: 43.
  • the present invention also includes non-naturally occurring HMGB B box polypeptides.
  • a non-naturally occurring HMGB B box polypeptide has at least 60%, at least 70%, 75%, 80%, 85%, or 90%, and preferably at least 95% sequence identity to the sequence of SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:43, as determined using the BLAST program and parameters described herein.
  • the HMGB B box consists of the sequence of SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO: 43, or the amino acid sequence in the corresponding region of an HMGB protein in a mammal.
  • the present invention is directed to a polypeptide comprising a vertebrate HMGB B box or a fragment thereof that has B box biological activity, or a non-naturally occurring HMGB B box but not comprising a full length HMG.
  • a "B Box fragment that has B box biological activity” or a "B box biologically active fragment” is meant a fragment of an HMGB B box that has the activity of an HMGB B box.
  • B box fragment is the fragment comprising the first 20 amino acids of the HMGBl B box (SEQ ID NO:44 or SEQ ID NO:45), as described herein.
  • B box fragments can be generated using standard molecular biology techniques and the function of the fragment can be assayed by determining if the fragment, when administered to a cell, inhibits an immune response from the cell, compared to a suitable control, for example, using methods described herein.
  • the HMGB B box polypeptide is a substantially pure, or substantially pure and isolated polypeptide that has been separated from components that naturally accompany it.
  • the polypeptide may be in an unpurified form, for example, in a cell, cell milieu, or cell extract.
  • the critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components.
  • HMGB A box is a mammalian HMGB A box, for example, a mammalian HMGBl A box, such as a human HMGBl A box provided herein as SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:36.
  • HMGBl A box having HMGB A box immunosuppressive activity are also included in the present invention.
  • non-naturally occurring HMGB A boxes can be created without undue experimentation, which would inhibit immune response by a vertebrate cell exposed to an antigen.
  • These non-naturally occurring functional A boxes can be created, by example, by aligning amino acid sequences of HMGB A boxes from different sources, and making one or more substitutions in one of the sequences at amino acid positions where the A boxes differ. The substitutions are preferably made using the same amino acid residue that occurs in the compared A box. Alternatively, a conservative substitution can be made from either of the residues.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains. For example, one grouping of amino acids includes those amino acids have neutral and hydrophobic side chains (a, v, 1, i, p, w, f, and m); another grouping is those amino acids having neutral and polar side chains (g, s, t, y, c, n, and q); another grouping is those amino acids having basic side chains (k, r, and h); another grouping is those amino acids having acidic side chains (d and e); another grouping is those amino acids having aliphatic side chains (g, a, v, 1, and i); another grouping is those amino acids having aliphatic-hydroxyl side chains (s and t); another grouping is those amino acids having amine-containing side chains (n, q, k, r, and h); another grouping is those amino acids having aromatic side chains (f,
  • the preferred conservative amino acid substitutions groups are: r-k; e-d, y-f, 1-m; v-i, and q-h. While a conservative amino acid substitution would be expected to preserve the biological activity of an HMGB A box polypeptide, the following is one example of how non-naturally occurring A box polypeptides can be made by comparing the human HMGBl A box (SEQ ID NO:4) with residues 32 to 85 of SEQ ID NO:3 of the human HMG2 A box (SEQ ID NO:56).
  • a non-naturally occurring HMGB A box can be created by, for example, by substituting the alanine (a) residue at the third position in the HMGBl A box with the serine (s) residue that occurs at the third position of the HMGB2 A box.
  • the substitution would provide a functional non- naturally occurring A box because the s residue functions at that position in the HJVIGB2 A box.
  • the third position of the HMGB 1 A box can be substituted with any amino acid that is conservative to alanine or serine, such as glycine (g), threonine (t), valine (v) or leucine (1).
  • g glycine
  • t threonine
  • v valine
  • leucine (1) leucine
  • any non-naturally occurring HMGB A box can be determined without undue experimentation by simply adding it to cells along with an antigen, and determine whether the A box inhibits an immune response by the cells, using, for example, methods described herein.
  • the cell from which the A box or an A box biologically active fragment can immunosuppress can be any cell that can be induced to produce an immune response.
  • the cell is an immune cell, for example, a macrophage, a monocyte, or a neutrophil.
  • Polypeptides comprising an A box or A box biologically active fragment that can inhibit immune activity, now known or later discovered, are within the scope of the present invention.
  • the present invention is directed to methods of treatment and inhibition of an immune pathology comprising administering a polypeptide comprising a vertebrate HMGB B box, or an immunosuppressive fragment thereof.
  • HMGB B boxes show a high degree of sequence conservation (see, for example, FIG. 3 for an amino acid sequence comparison of rat, mouse, and human HMGB polypeptides)
  • functional non-naturally occurring HMGB B boxes can be created without undue experimentation by making one or more conservative amino acid substitutions, or by comparing naturally occurring vertebrate B boxes from different sources and substituting analogous amino acids, as was discussed above with respect to the creation of functional non-naturally occurring A boxes.
  • the B box comprises SEQ ID NO:5, SEQ ID NO: 8, or SEQ ID NO:43, which are the sequences (three different lengths) of the human HMGBl B box, or is a fragment of an HMGB B box that has B box biological activity.
  • SEQ ID NO: 8 a 20 amino acid sequence contained within SEQ ID NO: 8 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:45).
  • Another example of an HMGB B box biologically active fragment consists of amino acids 1-20 of SEQ ID NO: 5 (napkrppsaf flfcseyrpk; SEQ ID NO:44).
  • the present invention provides a method of treating an immune pathology in an individual, or treating an individual at risk for having an immune pathology, comprising administering to the individual an effective amount of a HMGB polypeptide as described herein.
  • the HMGB polypeptide can be administered prior to, or concurrently with, or shortly thereafter, the antigen.
  • the polypeptide is administered to a patient prior to administration of the antigen.
  • the HMGB polypeptide can be administered alone, or in conjunction with the antigen administered for therapeutic purposes.
  • the term "in conjunction with,” indicates that the HMGB polypeptide is administered at about the same time as the antigen, as described below.
  • the HMGB polypeptide can be administered to the individual (animal) as part of a pharmaceutical composition comprising the polypeptide and a pharmaceutically acceptable carrier or excipient and, optionally, one or more additional therapeutic agents.
  • the HMGB polypeptide and antigen can be components of separate pharmaceutical compositions which can be mixed together prior to administration or administered separately.
  • the HMGB polypeptide can, for example, be administered in a composition containing the antigen, and thereby administered contemporaneously with the antigen.
  • the HMGB polypeptides and the antigen can be administered contemporaneously, without mixing (e.g., by delivery of the HMGB polypeptide on the intravenous line by which the antigen is also administered, or vice versa).
  • the HMGB polypeptide can be administered separately (e.g. , not admixed), but within a short time frame (e.g., within 24 hours) of administration of the antigen.
  • more than one HMGB polypeptide can be administered to the patient.
  • the HMGB polypeptide can be administered in an "effective amount" (i.e., a dosage amount that is sufficient to treat the immune pathology (disease), such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity, longevity or frequency of symptoms associated with the particular pathology or disease, as described herein).
  • an effective amount of the polypeptides of the invention is a quantity which will result in a therapeutic or prophylactic benefit for the animal.
  • the effective amount will vary, depending on such factors as the route of administration, the condition of the patient, the nature and extent of the disease's effects, and the like. Such factors are capable of determination by those skilled in the art.
  • the term "effective amount” also means the total amount of each active component of the composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • an effective amount of a HMGB polypeptide is an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, such as an amount sufficient to inhibit an immune response.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • an effective amount of the HMGB polypeptide is about 1 mg/kg of body weight to about 20 mg/kg of body weight of the individual.
  • the effective amount of the HMGB polypeptide is about 1-10 mg enzyme/kg body weight of the individual.
  • the effective amount of the HMGB polypeptide is about 1-5 mg enzyme/kg body weight of the individual.
  • an effective amount of 10 mg enzyme/kg body weight is administered. In another embodiment, an effective amount of 5 mg enzyme/kg body weight is administered.
  • the effective amount for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual.
  • the peptides are administered to a patient in need thereof in an amount sufficient to inhibit an immune response.
  • the terms "therapy,” “therapeutic,” and “treatment” as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, an immune disease or a detrimental immune condition, including preventing or delaying the onset of the disease symptoms, and/or lessening the severity or frequency of symptoms of the disease or condition.
  • subject and “individual” are defined herein to include animals such as mammals, including, but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species.
  • the animal is a human.
  • inhibit or “decrease” encompasses at least a small but measurable reduction in immune responses.
  • the immune response is inhibited by at least 20% over non-treated controls; in more preferred embodiments, the inhibition is at least 50%; in still more preferred embodiments, the inhibition is at least 70%, and in the most preferred embodiments, the inhibition is at least 80%.
  • the immune response is inhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, 75%, 80%, or 90% over non- treated controls. Inhibition can be assessed using methods described herein or other methods known in the art. Such reductions in immune response are capable of reducing the deleterious effects of an immune pathology in in vivo embodiments.
  • an detrimental immune response is inhibited by at least
  • the polypeptides can inhibit an autoimmune disorder.
  • autoimmune disorders which can be usefully treated using the present invention include those conditions enumerated in this specification.
  • the autoimmune disorder is ulcerative colitis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, Crohn's disease, asthma, allergy, and allograft rejection.
  • Autoimmune disorders are not included within the term "immune pathology" as the term is used herein.
  • An excipient and/or a carrier can be administered with the polypeptide.
  • the excipient and/or carrier is chosen based on the expected route of administration of the composition in therapeutic applications.
  • the route of administration of the polypeptide depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder, and oral administration may be preferred to treat a gastrointestinal disorder.
  • the route of administration and the dosage of the composition or polypeptide to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the polypeptide can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabuccally and transdermally to the patient.
  • compositions comprising polypeptides of the present invention designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier.
  • the polypeptide may be enclosed in gelatin capsules or compressed into tablets.
  • the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
  • Tablets, pills, capsules, troches and the like may also contain binders, excipients, disintegrating agents, lubricants, sweetening agents, and flavoring agents.
  • binders include microcrystalline cellulose, gum tragacanth and gelatin.
  • excipients include starch or lactose.
  • disintegrating agents include alginic acid, corn starch and the like.
  • lubricants include magnesium stearate or potassium stearate.
  • glidant is colloidal silicon dioxide.
  • sweetening agents include sucrose, saccharin and the like.
  • flavoring agents include peppermint, methyl salicylate, orange flavoring and the like.
  • Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • the polypeptides of the present invention can be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the polypeptides and compositions of the present invention into a solution or suspension.
  • Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • Parenteral formulations may also include antibacterial agents such as, for example, benzyl alcohol or methyl parabens, antioxidants such as, for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added.
  • the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
  • Rectal administration includes administering the polypeptide into the rectum or large intestine. This can be accomplished using suppositories or enemas.
  • Suppository formulations can be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120°C, dissolving the polypeptide in the glycerin, mixing the heated glycerin (after which purified water may be added), and pouring the hot mixture into a suppository mold.
  • Transdermal administration includes percutaneous absorption of the polypeptide through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like.
  • the present invention also includes nasally administering to the mammal a ' therapeutically effective amount of the polypeptide.
  • nasally administering or nasal administration includes administering the polypeptide to the mucous membranes of the nasal passage or nasal cavity of the patient.
  • Pharmaceutical compositions for nasal administration of a polypeptide include therapeutically effective amounts of the polypeptide prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the polypeptide may also take place using a nasal tampon or nasal sponge.
  • the adjuvant and polypeptide are administered at the same site.
  • the polypeptides described herein can also include one or more additional agents used to treat an immune pathology.
  • the agent may be, for example, an antagonist of an early immune response mediator.
  • an effective amount can range from 0.01 mg per day to about 100 mg per day for an adult.
  • the dosage ranges from about 1 mg per day to about 100 mg per day or from about 1 mg per day to about 10 mg per day.
  • the polypeptide or fragment can be administered by providing a DNA or RNA vector encoding the polypeptide e.g., a B box or B box fragment, with the appropriate control sequences operably linked to the nucleic acid encoding the polypeptide, so that the polypeptide is synthesized in the treated cell or patient.
  • Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the invention will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples and claims, be considered exemplary only.
  • Example 1 Materials and Methods Cloning of HMGBl and Production of HMGBl Mutants The following methods were used to prepare clones and mutants of human HMGBl.
  • Recombinant full length human HMGBl (HOLO-HMG, also known as Ml) (651 base pairs; GenBank Accession Number U51677) was cloned by PCR amplification from a human brain Quick-Clone cDNA preparation (Clontech, Palo Alto, CA) using the following primers; forward primer: 5' GATGGGCAAAGGAGATCC TAAG 3' (SEQ TD NO:46) and reverse primer: 5' GCGGCCGCTTATTCATCA TCATCATCTTC 3' (SEQ ID NO:47) (see FIG. 1).
  • Human HMGBl mutants were cloned and purified as follows. Truncated forms of human HMGBl was cloned by PCR amplification from a Human Brain Quick- Clone cDNA preparation (Clontech, Palo Alto, CA). The primers used were (forward and reverse, respectively):
  • Carboxyl terminus mutant also known as nAB or M2
  • nAB or M2 Carboxyl terminus mutant
  • 557 bp 5' GATGGGCAAAGGAGATCCTAAG 3' (SEQ ID NO:48) and 5' GCGGCCGC TCACTTGCTTTTTTCAGCCTTGAC 3' (SEQ ID NO:49)
  • B box mutant (233 bp): 5' AAGTTCAAGGATCCCAATGCAAAG 3' (SEQ ID NO:52) and 5' GCGGCCGCTCAATATGCAGCTATATCCTTTTC 3' (SEQ ID NO:53)
  • Amino terminus+A box mutant (261 bp): 5' GATGGGCAAAGGAGATCCTAAG 3' (SEQ ID NO:54) and 5' TCACTTTTTTGTCTCCCCTTTGGG 3' (SEQ ID NO:55).
  • a stop codon was added to each mutant to ensure the accuracy of protein size.
  • PCR products were subcloned into pCRII-TOPO vector EcoRI sites using the TA cloning method per manufacturer's instruction (Invitrogen, Carlsbad, CA). After amplification, the PCR product was digested with EcoRI and subcloned onto expression vector with a GST tag pGEX (Pharmacia); correct orientation and positive clones were confirmed by DNA sequencing on both strands.
  • HMGB mutants generated as described above have the following amino acid sequences:
  • Peptide Synthesis Peptides were synthesized and HPLC purified at Utah State University Biotechnology Center (Logan, Utah) at 90% purity. Statistical Analysis Data are presented as mean ⁇ SEM unless otherwise stated. Differences between groups were determined by two-tailed Student's t-test, one-way ANOVA followed by the least significant difference test or 2 tailed Fisher's Exact Test.
  • HMGBl has 2 folded DNA binding domains (A and B boxes) and a negatively charged acidic carboxyl tail.
  • a and B boxes DNA binding domains
  • a negatively charged acidic carboxyl tail To elucidate the structural basis of HMGBl cytokine activity, full length and truncated forms of HMGBl were expressed by mutagenesis and the purified proteins were screened for stimulating activity in monocyte cultures (FIG. 1).
  • Full length HMGBl, a mutant in which the carboxyl terminus was deleted (resulting in an HMGB truncated at the carboxyl terminus, nAB), a mutant containing only the B box, and a mutant containing only the A box were generated.
  • mutants of human HMGBl were made by polymerase chain reaction (PCR) using specific primers as described herein, and the mutant proteins were expressed using a glutathione S-transferase (GST) gene fusion system (Pharmacia Biotech, Piscataway, NJ) in accordance with the manufacturer's instructions. Briefly, DNA fragments, made by PCR methods, were fused to GST fusion vectors and amplified in E. coli. The expressed HMGBl protein and HMGB 1 mutants and were then isolated using GST affinity column.
  • GST glutathione S-transferase
  • Example 2 Inhibition of Splenocyte Mitogenicity The assay was performed essentially as described in Hellman J, Roberts JD, Tehan MM, Allaire J, Warren HS, "Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in Gram-negative sepsis and causes inflammation and death in mice," J. Biol. Chem., 277(16):14274-80 (2002)).
  • Splenocytes were prepared from LPS hyporesistant mice (C3H/HeJ mice) and were maintained in RPMI 1640 (Mediatech, Cellgro) supplemented with L-glutamine, 5% heat- inactivated FCS, and gentamicin 50 ⁇ g/ml as described in Coligan JE, Kruisbeck AM, Margulies DH, Shervach EM and Strober W: Current Protocols in Immunology, John Wiley and Sons, New York (2001)).
  • HEMT Hemaize calf serum
  • PAL peptidoglycan-associated lipoprotein
  • LPS lipopolysaccharide
  • the HMGB fragments include A (A box plus amino terminus), B (B box at 2.5 ⁇ g/ml), AB (A box plus B box), B10 (B box at 10 ⁇ g/ml), nAB (A box plus B box plus amino terminus), and AB (A box plus B box).
  • the cells were harvested (PHD Cell Harvester, Cambridge Technology, Inc) and 3 H-thymidine incorporation into DNA was assessed by liquid scintillation counting. (See FIG. 5).
  • Example 3 Inhibition of Antibody Response Blood was obtained from groups of 10-13 C3H/HeJ mice (20-25 grams).
  • Ovalbumin 40 ⁇ g
  • PAL peptidoglycan-associated lipoprotein
  • nAB recombinant fragment of HMGB-1 truncated at the carboxyl terminus
  • Sera were tested for anti-ovalbumin IgG by ELISA, in a manner similar as that described previously only using ovalbumin as the antigen on the microtiter plate (Siber GR, Kania S, Warren HS. "Cross reactivity of rabbit antibodies to lipopolysaccharides of Escherichia coli J5 and other gram-negative bacteria," J. Infect. Dis., 152:954-964 (1985). (see FIG. 7 and FIG. 8) Results were tested for significance by unpaired two-tailed T test using Graphpad software. (See FIG. 8). A similar set of experiments was utilized to study the induction of antibody in the presence of the adjuvant PAL.
  • mice Groups of 4 mice were injected in the right thigh muscle with PAL (10 ⁇ g) together with tetanus toxoid (TT, 10 ⁇ g) or tetanus toxoid (10 ⁇ g) and nAB (10 ⁇ g) at time points zero, 2 weeks and four weeks. Blood was obtained from the mice prior to each vaccination and at 6 weeks. Sera were tested for the presence of anti-tetanus IgG by ELISA as above. (See FIG. 9). While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Abstract

La présente invention concerne des procédés pour traiter une pathologie immunitaire chez un individu, comprenant l'administration audit individu d'une quantité efficace d'un polypeptide protéine du domaine du groupe à mobilité élevée (high mobility group box protein / HMGB), comprenant un domaine de HMGB A de vertébré, un domaine de HMGB A d'origine artificielle, ou un fragment immunosuppresseur dudit domaine de HMGB1 A de vertébré ou du domaine de HMGB1 A d'origine artificielle.
PCT/US2004/029540 2003-09-10 2004-09-10 Utilisation de hmgb et fragments de hmgb pour faire diminuer la reponse immunitaire specifique WO2005025604A2 (fr)

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WO2006024547A3 (fr) * 2004-09-03 2006-06-01 Creabilis Therapeutics Spa Utilisation de peptides obtenus par mutations systematiques d'aminoacides simples de la sequence a humaine ou non-humaine de la hmgb 1 pour prevenir et/ou antagoniser des pathologies induites par la hmgb 1
WO2007031100A1 (fr) * 2005-09-14 2007-03-22 Ostini, Marco Immunothérapie active pour inflammation systémique potentiellement mortelle
WO2008053892A1 (fr) * 2006-10-30 2008-05-08 Genomix Co., Ltd. Produit pharmaceutique servant à favoriser la régénération fonctionnelle d'un tissu endommagé
EP2039367A1 (fr) * 2006-06-01 2009-03-25 National Universiy Corporation Tokyo Medical and Dental University Agent prophylactique/thérapeutique pour maladie neurodégénérative
WO2012034090A1 (fr) * 2010-09-09 2012-03-15 University Of Southern California Compositions et procédés pour éliminer des biofilms
US8673580B2 (en) 2008-04-30 2014-03-18 Genomix Co., Ltd. Agent for recruitment of bone-marrow-derived pluripotent stem cell into peripheral circulation
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US9623078B2 (en) 2012-10-25 2017-04-18 Genomix Co., Ltd. Method for treating cardiac infarction using HMGB1 fragment
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WO2019107530A1 (fr) * 2017-12-01 2019-06-06 株式会社ステムリム Agent thérapeutique pour une maladie inflammatoire de l'intestin
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US11248040B2 (en) 2013-09-26 2022-02-15 Trellis Bioscience, Llc Binding moieties for biofilm remediation
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092004A2 (fr) * 2001-05-15 2002-11-21 North Shore-Long Island Jewish Research Institute Utilisation de fragments de hmg en tant qu'agents anti-inflammatoires
WO2004046345A2 (fr) * 2002-11-20 2004-06-03 Critical Therapeutics, Inc. Utilisation de fragments hmgb en tant qu'agents anti-inflammatoires
WO2004046338A2 (fr) * 2002-11-20 2004-06-03 North Shore-Long Island Jewish Research Institute Utilisation de polypeptides hmgb permettant d'accroitre des reponses immunitaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092004A2 (fr) * 2001-05-15 2002-11-21 North Shore-Long Island Jewish Research Institute Utilisation de fragments de hmg en tant qu'agents anti-inflammatoires
WO2004046345A2 (fr) * 2002-11-20 2004-06-03 Critical Therapeutics, Inc. Utilisation de fragments hmgb en tant qu'agents anti-inflammatoires
WO2004046338A2 (fr) * 2002-11-20 2004-06-03 North Shore-Long Island Jewish Research Institute Utilisation de polypeptides hmgb permettant d'accroitre des reponses immunitaires

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WO2006024547A3 (fr) * 2004-09-03 2006-06-01 Creabilis Therapeutics Spa Utilisation de peptides obtenus par mutations systematiques d'aminoacides simples de la sequence a humaine ou non-humaine de la hmgb 1 pour prevenir et/ou antagoniser des pathologies induites par la hmgb 1
US8058232B2 (en) 2004-09-03 2011-11-15 Creabilis Therapeutics S.P.A. HMGB1 high affinity binding domain Box-A mutants
WO2007031100A1 (fr) * 2005-09-14 2007-03-22 Ostini, Marco Immunothérapie active pour inflammation systémique potentiellement mortelle
US7833975B2 (en) 2006-06-01 2010-11-16 National University Corporation Tokyo Medical And Dental University Prophylactic/therapeutic agent for neurodegenerative disease
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US8673580B2 (en) 2008-04-30 2014-03-18 Genomix Co., Ltd. Agent for recruitment of bone-marrow-derived pluripotent stem cell into peripheral circulation
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US8961990B2 (en) 2010-06-09 2015-02-24 The Board Of Trustees Of The University Of Arkansas Vaccine and methods to reduce campylobacter infection
US10960068B2 (en) 2010-06-09 2021-03-30 The Board Of Trustees Of The University Of Arkansas Vaccine and methods to reduce campylobacter infection
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