WO1997039121A1 - Peptides du recepteur de produits terminaux d'une glycosylation avancee, et utilisation de ces peptides - Google Patents

Peptides du recepteur de produits terminaux d'une glycosylation avancee, et utilisation de ces peptides Download PDF

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WO1997039121A1
WO1997039121A1 PCT/EP1997/001832 EP9701832W WO9739121A1 WO 1997039121 A1 WO1997039121 A1 WO 1997039121A1 EP 9701832 W EP9701832 W EP 9701832W WO 9739121 A1 WO9739121 A1 WO 9739121A1
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seq
rage
age
polypeptide
acid sequence
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PCT/EP1997/001832
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Michael John Morser
Mariko Nagashima
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Schering Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • AGEs Advanced glycosylation end-products of proteins
  • AGEs are nonenzymaticaliy glycosylated proteins which have been shown to accumulate in vascular tissue in aging and at an accelerated rate in individuals with diabetes.
  • AGEs result from the non-enzymatic, but concentration dependant interaction of glucose and other reducing sugars with amino groups on proteins to form glycosylated proteins termed Amadori adducts. Over time, these Amadori adducts undergo additional rearrangements, dehydrations and cross-linking with other proteins to accumulate as a family of complex structures referred to as AGEs.
  • AGEs have been shown to bind specifically in a saturable and reversible manner to cell surface receptors, including receptors expressed on the surface of endothelial cells and particularly those of the microvasculature, monocytes/macrophages, smooth muscle cells, mesengial cells and neurons. Following binding to cell surface receptors, AGEs are taken up in vesicles and either degraded intracellulariy or transported through the cells and deposited in the sub-endothelial matrix, where they accumulate. Esposito et al., J. Exp. Med. 170:1387-1407 (1989). In addition, chemotactic signals for monocytes, but not other white cells are released. These monocytes then adhere and diapedese through the endothelial cell layer. Kirstein et al., Proc. Nat'l Acad. Sci. USA 187:9010-9014 (1990).
  • AGEs also have been shown to cause proliferation of endothelial cells, which become more permeable and more thrombogenic, i.e., thrombomodulin is downregulated while tissue factor is upregulated. Esposito et al., J. Exp. Med. 170:1387-1407 (1989).
  • Monocytes ⁇ macrophages can also take up AGEs through their receptors which are distinct from the acetyl-LDL receptors, but which may be related to the receptors for aldehyde-modified proteins. Takata et al., J. Biol. Chem. 263:14819-14825 (1988), Takata et al., Biochim. Biophys. Acta 986:18-26 (1989), Radoff et al., Diabetes 39:1510-1518 (1990). Binding of AGEs to monocytes in vitro leads to the induction of cytokines, TNF and IL-1 , which then stimulate the release of a number of other growth factors responsible for cell proliferation, migration and matrix synthesis.
  • the present invention provides substantially pure polypeptides which comprise a biologically active, soluble, human RAGE polypeptide.
  • the present invention also provides pharmaceutical compositions which present the polypeptides of the invention with a pharmaceutically acceptable carrier.
  • the polypeptides of the invention may be used in a number of applications.
  • the polypeptides may be used in methods for purifying ligands to RAGE, e.g., AGEs.
  • the methods generally comprise the steps of immobilizing the soluble human RAGE polypeptide on a solid support, followed by contacting the immobilized RAGE polypeptide with a mixture of proteins containing an AGE whereby the RAGE polypeptide selectively binds the AGE in the mixture of proteins.
  • the immobilized RAGE polypeptide is then washed to remove unbound proteins and the purified AGE which was selectively bound to the immobilized RAGE is eluted.
  • the invention provides models of AGE/AGE receptor interactions to identify effectors of this interaction.
  • the invention provides a method of screening a test compound to determine if that compound is an agonist or antagonist of a AGE/AGE receptor interaction. The method comprises separately incubating a soluble human RAGE polypeptide with o an AGE in the presence and absence of the test compound. The level of interaction between the soluble human RAGE polypeptide and the AGE in the presence and absence of the test compound is then detected. The level of interaction in the presence and absence of the test compound is then compared. An increase or decrease in the level of interaction in the presence of the test compound is indicative that the test compound is an agonist or antagonist of the interaction, respectively.
  • the present invention provides a method of inhibiting an interaction between an AGE and a receptor which specifically binds AGE, by contacting the AGE with an effective amount of a soluble human RAGE polypeptide.
  • the present invention provides methods of treating patients for symptoms of a disorder which are caused by an interaction between an AGE and its receptor.
  • the method comprises administering an effective amount of a soluble human RAGE polypeptide to the patient.
  • the present invention provides nucleic acid sequences which encode soluble human RAGE polypeptides.
  • the present invention provides the use of an effective amount of a soluble human RAGE polypeptide for the production of a pharmaceutical compound or composition for the treatment of a patient for symtoms of a disorder, wherein said symptoms are caused by an interaction between an AGE and its receptor.
  • said patients are diabetic patients, and said symptom is increased vascular permeability.
  • said disorder is Diabetes Mellitus and said symptom is selected from the group consisting of diabetic microvasculopathy, diabetic macrovasculopathy and occlusive vascular disorder.
  • said symptoms are selected from neuropathy, nephropathy, retinopathy and atherosclerosis.
  • said symptom is hemodialysis-associated amyloidosis.
  • Figures 1A and 1B show the nucleic acid sequence and deduced amino acid sequence of a soluble human RAGE polypeptide (Seq ID NOS: 1-4) .
  • Figure 1 A shows the DNA and amino acid sequence of soluble human RAGE which includes an expressed pre-sequence (Seq ID NOS: 1-2)
  • Figure 1 B shows the sequences for mature soluble human RAGE (Seq ID NOS: 3-4) .
  • Standard three-letter abbreviations are used to denote the individual amino acids.
  • Figures 2A and 2B show binding of an AGE-BSA protein to indirectly immobilized RAGE polypeptide.
  • Figure 2A shows total binding of labeled AGE-BSA to RAGE immobilized indirectly upon a solid support through a FLAG peptide/antiFLAG antibody interaction which mimics the cell surface presentation of RAGE (squares). Also shown is nonspecific binding (diamonds)(labeled AGE-BSA binding in the presence of excess unlabeled AGE-BSA).
  • Figure 2B shows corrected, specific binding of AGE-BSA to RAGE polypeptide that is indirectly coupled to the solid support.
  • Figure 3 shows a dose response curve for RAGE/AGE binding in the presence of increasing concentrations of free RAGE
  • Figure 4A is a bar graph showing levels of antibody binding to human soluble RAGE using an EIA antibody capture assay. Binding is compared between intact soluble RAGE (dark bars) and RAGE/DCC chimeric protein (in which the first Ig-like domain of RAGE is replaced with the first ig-like domain of DCC, a member of the lg superfamily.
  • Four antibodies (#5, 9, 14 and 19) recognized both the intact soluble RAGE and the chimeric protein.
  • Figures 4B and 4C show antibody binding to peptide fragments of soluble human RAGE, designated peptide 1 (CKGAPKKPPQ) (Seq ID NO: 5), fragment 2 (WKLNTGRTEAC) (Seq ID NO: 6) and fragment 8 (GPQDQGTYSC) (Seq ID NO: 7).
  • Figure 5 shows a Western Blot hybridization of anti-RAGE MAb SW1 E8 (ATCC Accession No. HB-12166). Lanes 1-3 represent rat, mouse and human RAGE expressed in Baculovirus, respectively. Lanes were loaded with 5 ⁇ l of conditioned media. Monomeric RAGE is apparent as a doublet at approximately 41 Kd.
  • Figures 6A and 6B show results of a direct antigen capture EIA.
  • Figure 6A shows a capture assay employing MAb RBF9D9 (ATCC Accession No. HB-12165) as the RAGE capture antibody and MAb SW10C1 (ATCC Accession No. HB-12164) as the RAGE detection antibody, which recognizes human, but not murine RAGE.
  • Figure 6B shows a capture assay employing MAb RBF9D9 (ATCC Accession No. HB-12165) as the RAGE capture antibody and MAb SW1E8 (ATCC Accession No. HB-12166) as the detection antibody, which recognizes both human and murine RAGE.
  • Figure 7 shows the results of flow cytometry of CHO parental cells and CHO cells transfected with full length human RAGE, using anti-RAGE MAb
  • SW10C1 ATCC Accession No. HB-12164. Sixty nine of the original 72 MAb panel demonstrated similar reactivity with cell surface RAGE.
  • Figures 8A and 8B show results of flow cytometry of CHO parental and CHO-RAGE transfectants incubated with lactoferrin and immunostained with anti-RAGE/phycoerythrin ( Figure 8A) and anti-lactoferrin/FITC ( Figure 8B).
  • Figures 9A and 9B show the in vitro permeability of confluent BEAC layers by albumin and inulin. Permeability is shown following incubation with medium (control, white bar), normal RBCs (black bar) and diabetic RBCs (hatched bars). Reversal of diabetic RBC associated permeability is demonstrated following pretreatment with soluble, recombinant RAGE (sRAGE) but not control protein (sVCAM).
  • sRAGE soluble, recombinant RAGE
  • sVCAM control protein
  • Figure 10 shows plasma pharmacokinetics of 125 l-huma ⁇ recombinant RAGE after an intravenous infusion into rat.
  • Figures 11A and 11B show comparisons of efficiency of recombinant soluble human RAGE and recombinant rat-VCAM in reversing permeability induced by diabetic RBC incubation in various tissues of normal ( Figure 11 A) and diabetic rats ( Figure 11B).
  • Figure 12 shows a schematic illustration of the blood-tissue albumin transport tracer uptake method. Also shown are the calculations used to obtain albumin clearance values.
  • Figures 13A, 13B and 13C show albumin clearance from various tissues in control rats (white bar), diabetic rats (left black bar), diabetic rats with soluble RAGE pretreatment (middle black bar) and diabetic rats with soluble VCAM- 1 pretreatment (right black bar) in early ( Figure 13A), mid ( Figure 13B) and late (Figure 13C) phases of STZ-induced diabetes.
  • Figures 14A, 14B and 14C show extravascular water levels from various tissues in control rats (white bar), diabetic rats (left black bar), diabetic rats with soluble RAGE pretreatment (middle black bar) and diabetic rats with soluble VCAM-1 pretreatment (right black bar) in early ( Figure 14A), mid ( Figure 14B) and late (Figure 14C) phases of diabetes.
  • Figures 15A and 15B show adhesion of RAGE pretreated and control RBCs to single vessels. DETAILED DESCRIPTION OF THE INVENTION I. General
  • AGE formation in biological systems is dependant upon blood-glucose concentration and time of incubation. Without being bound to a particular theory, it is believed that this time/concentration dependency accounts for the adverse effects in hyperglycemic diabetics as well as the elderly. Similarly, proteins which themselves have a longer half-life, are more prone to undergo glycosylation to form AGEs. In diabetic patients, a high level of plasma glucose leads to glycosylation of various plasma proteins, including hemoglobin and LDL (low-density lipoprotein) as well as enzymes and matrix proteins.
  • AGEs induce a number of permanent abnormalities in the extracellular matrix component function, and stimulate cytokines and reactive oxygen species production through AGE-specific receptors. Inhibition of AGE formation in long term diabetic animals has also been shown to prevent or reduce the severity of a number of elements of the pathology of diabetes, including retinopathy, nephropathy, neuropathy and arterial abnormalities. Brownlee, Ann. Rev. Med. 46:223-234 (1995), Zimmerman et al., Proc. Nat'l Acad. Sci. USA 92:3744-3748 (1995).
  • a number of proteins associated with Alzheimer's disease e.g., amyloid, tau, the major components of neurofibrillary tangles and senile plaques, are found to be similarly modified.
  • Smith et al. Nature 374:316 (1995), Smith et al.,Proc. Nat'l Acad. Sci. USA, 91 :5710-5714 (1994), Vitek et al., Proc. Nat'l Acad. Sci. USA, 91:4766-4770 (1994).
  • ⁇ 2 -microglobulin a major component of amyloid fibrils, is modified by glycosylation. Miyata et al., J. Clin.
  • compositions may be used in a variety of applications including therapeutic applications, e.g., as blocking agents to inhibit or otherwise reduce the AGE/RAGE interaction, screening applications, e.g., as models of the AGE/RAGE interaction, and diagnostic applications, e.g., to identify abnormal levels of AGE or RAGE in a given system.
  • therapeutic applications e.g., as blocking agents to inhibit or otherwise reduce the AGE/RAGE interaction
  • screening applications e.g., as models of the AGE/RAGE interaction
  • diagnostic applications e.g., to identify abnormal levels of AGE or RAGE in a given system.
  • the present invention provides compositions comprising soluble RAGE polypeptides, antibodies that are specifically immunoreactive with soluble RAGE polypeptides, and methods of using these compositions in screening, therapeutic and diagnostic applications.
  • the present invention provides substantially pure or isolated polypeptides that are related to and/or derived from human RAGE polypeptides.
  • substantially pure or isolated when referring to proteins and polypeptides, denote those polypeptides that are separated from proteins or other contaminants with which they are naturally associated.
  • a protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure or isolated protein or polypeptide will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.
  • the isolated polypeptides of the present invention are related to and/or derived from soluble human RAGE polypeptides.
  • soluble generally refers to RAGE derived polypeptides that lack a transmembrane region that is associated with full length RAGE polypeptides.
  • soluble RAGE polypeptides generally comprise fragments of the extracellular domain of RAGE.
  • the soluble peptides of the invention will comprise one or more of the Ig-like domains of the extracellular region of RAGE.
  • AGE refers to an advanced glycosylation end- product. Typically, such AGEs may be full length proteins, polypeptides or aggregations of proteins and/or polypeptides.
  • polypeptides of the invention also may be characterized by their ability to either mimic or inhibit the interaction between AGEs and their receptors, e.g., RAGE.
  • AGE or AGE receptor "mimics” Those polypeptides which are mimetic of either AGE or its receptors in the AGE/receptor interaction are termed AGE or AGE receptor "mimics".
  • polypeptides of the invention will have an amino acid sequence that is related to or derived from the amino acid sequence of soluble human RAGE as shown in Figures 1A and 1B (Seq ID NOS: 1- 4). Although described in terms of the amino acid sequence shown in Figure 1A and 1B (Seq ID NOS: 1-4), it will be readily understood that the polypeptides of the present invention include those peptides having the listed amino acid sequence or biologically active fragments thereof, as well as those polypeptides having amino acid sequences that are substantially homologous to the listed sequence.
  • substantially homologous when referring to polypeptides, refer comparatively to two amino acid sequences which, when optimally aligned, are at least about 75% homologous, preferably at least about 85% homologous more preferably at least about 90% homologous, and still more preferably at least about 95% homologous.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • biologically active fragment refers to portions of the soluble RAGE polypeptides, which possess a particular biological activity.
  • biological activity may include the ability to bind a particular protein, substrate or ligand, e.g., AGE, to have antibodies generated against it, to block or otherwise inhibit an interaction between two proteins, e.g., a receptor and its ligand, such as AGE and RAGE, between an enzyme and its substrate, between an epitope and an antibody, or such fragments may include a particular catalytic activity.
  • polypeptides of the present invention particularly preferred polypeptides or biologically active fragments include, e.g., polypeptides that possess one or more of the biological activities described above, such as the ability to specifically interact with AGEs, the ability to block, reduce, or otherwise inhibit the interaction between AGEs and RAGE, and the ability to elicit antibodies that are specifically immunoreactive with AGEs or RAGE.
  • Those fragments that are specifically recognized and bound by antibodies raised against the polypeptides of the invention are also included in the definition of biologically active fragments.
  • immunologically active fragments include those fragments comprising the amino acid sequences specifically described below, and particularly those selected from the group consisting of: WKLNTGRTEA (Seq ID NO: 8), CEVPAQPSPQI (Seq ID NO: 9), CRAMNQNGKETKSN (Seq ID NO: 10), GPQDQGTYSC (Seq ID NO: 11), AQNITARIGEPLVLK (Seq ID NO: 12), CKGAPKKPPQ (Seq ID NO: 13), EQTRRHPET (Seq ID NO: 14), RGGDPRPTFSC (Seq ID NO: 15), SPGLPRHRAL (Seq ID NO: 16), and SSHGPQESRA (Seq ID NO: 17).
  • polypeptides of the invention may further include modifications to the N- or C-termini, i.e., acetylation, amidation, or inclusion of additional amino acids, i.e., cysteine, to assist in conjugation with other proteins or compounds, e.g., polypeptides having the following sequences: WKLNTGRTEAC (Seq ID NO: 6); AQNITARIGEPLVLKC (Seq ID NO: 18); CEQTRRHPET (Seq ID NO: 19); CSPGLPRHRAL (Seq ID NO: 20); and SSHGPQESRAC (Seq ID NO: 21).
  • WKLNTGRTEAC Seq ID NO: 6
  • AQNITARIGEPLVLKC Seq ID NO: 18
  • CEQTRRHPET Seq ID NO: 19
  • CSPGLPRHRAL Seq ID NO: 20
  • SSHGPQESRAC Seq ID NO: 21
  • polypeptides of the invention may also be characterized by their ability to block the interaction between two proteins, e.g. AGE and RAGE, RAGE and anti-RAGE Abs, or AGE and anti-AGE Abs.
  • AGE and RAGE e.g. AGE and RAGE
  • RAGE and anti-RAGE Abs e.g. AGE and anti-RAGE Abs
  • AGE and anti-AGE Abs e.g. AGE and anti-AGE Abs
  • AGE and anti-AGE Abs e.g., AGE and anti-RAGE Abs.
  • polypeptides of the present invention may also be characterized by their ability to bind antibodies raised against proteins or polypeptides having the amino acid sequences of soluble human RAGE, as shown in Figure 1A and 1B (Seq ID NOS: 1-4) , or fragments thereof. These antibodies generally recognize polypeptides that are homologous to at least portions of human RAGE proteins or their immunologically active fragments.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or domain. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
  • the biologically active fragments of the polypeptides described herein may include any subsequence of the above described RAGE polypeptide. Typically, however, such biologically active fragments will range in size from about 10 amino acids in length to about 320 amino acids in length. More typically, the biologically active fragments will be from about 10, 11 , 12, 13, 14, 15 or 16 amino acids in length to about 50 amino acids in length.
  • Examples of particularly preferred biologically active fragments of soluble human RAGE include peptides comprising an amino acid sequence selected from the group consisting of WKLNTGRTEA (Seq ID NO: 8), CEVPAQPSPQI (Seq ID NO: 9), CRAMNQNGKETKSN (Seq ID NO: 10), GPQDQGTYSC (Seq ID NO: 11), AQNITARIGEPLVLK (Seq ID NO: 12),
  • polypeptides of the present invention may generally be prepared using recombinant or synthetic methods that are well known in the art.
  • polypeptides of the present invention may be expressed by a suitable host cell that has been transfected with a nucleic acid of the invention, as described in greater detail below.
  • Biologically active fragments of the above described polypeptides may generally be identified and prepared using methods well known in the art. For example, selective proteolytic digestion, recombinant deletional methods or de novo peptide synthesis methods may be employed to identify portions of the above described peptides that possess the desired biological activity, e.g., AGE binding, presence of immunological determinants, and the like. See, e.g., Sambrook, et al. Isolation and purification of the polypeptides of the present invention can be carried out by methods that are generally well known in the art.
  • the polypeptides may be purified using readily available chromatographic methods, e.g., ion exchange, hydrophobic interaction, HPLC or affinity chromatography, to achieve the desired purity. Affinity chromatography may be particularly attractive in allowing an individual to take advantage of the specific biological activity of the desired peptide, e.g., AGE binding, presence of antigenic determinants or the like.
  • antibodies raised against human RAGE polypeptides or its immunologically active fragments may be coupled to a suitable solid support and contacted with a mixture of proteins containing the polypeptides of the invention under conditions conducive to the association of these polypeptides with the antibody.
  • the solid support is washed to remove unbound material and/or nonspecifically bound proteins.
  • the desired polypeptides may then be eluted from the solid support in substantially pure form by, e.g., a change in salt, pH or buffer concentration.
  • the affinity of the soluble RAGE polypeptides for AGEs may be used advantageously to purify these peptides.
  • AGEs e.g. BSA-AGE, may be immobilized as described above, for use as affinity probes in the purification of the soluble RAGE polypeptides.
  • the present invention also provides fusion proteins which contain these polypeptides or fragments. Fusion proteins may be useful in providing for enhanced expression of the RAGE polypeptide constructs, or in producing RAGE polypeptides having other desirable properties, e.g., labeling groups, e.g., enzymatic reporter groups, binding groups, antibody epitopes, etc.
  • the term "fusion protein" as used herein generally refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two distinct, heterologous polypeptides which are not normally fused together in a single amino acid sequence.
  • a fusion protein may include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in a single amino acid sequence.
  • Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a gene fusion, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.
  • amino acid variants of the above described polypeptides may include insertions, deletions and substitutions with other amino acids.
  • conservative amino acid substitutions may be made, i.e., substitution of selected amino acids with different amino acids having similar structural characteristics, e.g., net charge, hydrophobicity and the like.
  • conservative substitutions include, e.g., Ala:Val:Leu:lle:Met, Asp:Glu, Lys:Arg, Asn:Gln, Phe.Tyr and Ser:Thr Glycosylation modifications, either changed, increased amounts or decreased amounts, as well as other sequence modifications are also included within the polypeptides of the invention.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may also be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61:387; for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • modification of the amino or carboxy terminals may also be used to confer stabilizing properties upon the polypeptides of the invention, e.g., amidation of the carboxy-terminus or acylation of the amino-terminus.
  • substitution of amino acids involved in catalytic activity can be used to generate dominant negative inhibitors of signaling pathways.
  • polypeptides one of skill in the art, upon reading the instant specification, will appreciate that these terms also include structural analogs and derivatives of the above-described polypeptides, e.g., polypeptides having conservative amino acid insertions, deletions or substitutions, peptidomimetics, and the like.
  • polypeptides which consist only of naturally- occurring amino acids
  • peptidomimetics of the polypeptides of the present invention are also provided.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and
  • Peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production; greater chemical stability; enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.); altered specificity (e.g., a broad-spectrum of biological activities); reduced antigenicity; and others.
  • detectable groups may comprise a detectable protein group, e.g., an assayable enzyme or antibody epitope as described above in the discussion of fusion proteins.
  • the detectable group may be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., 125 l, 32 P or 35 S) or a chemiluminescent or fluorescent group.
  • the detectable group may be a substrate, cofactor, inhibitor or affinity ligand.
  • Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • non-interfering positions generally are positions that do not form direct contacts with the molecules to which the peptidomimetic binds (e.g., AGE) to produce the therapeutic effect.
  • Derivitization (e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • peptidomimetics of peptides of the invention bind to their ligands (e.g., AGEs) with high affinity and/or possess detectable biological activity (i.e., are agonistic or antagonistic to AGE/RAGE interaction and phenotypic changes brought about by those interactions).
  • ligands e.g., AGEs
  • detectable biological activity i.e., are agonistic or antagonistic to AGE/RAGE interaction and phenotypic changes brought about by those interactions.
  • the present invention provides antibodies that are specifically immunoreactive with human RAGE and more particularly, the soluble human RAGE polypeptides of the invention.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Hariow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • an appropriate target immune system is selected, typically a mouse or rabbit, but also including goats, sheep, cows, guinea pigs, monkeys and rats.
  • the substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal. These and other parameters are well known to immunologists.
  • injections are given in the footpads, intramuscularly, intradermaily or intraperitoneaUy.
  • the immunoglobulins produced by the host can be precipitated, isolated and purified by routine methods, including affinity purification.
  • spleens of these animals are excised and individual spleen cells are fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone are tested for the production of an appropriate antibody specific for the desired region of the antigen.
  • Techniques for producing antibodies are well known in the art. See, e.g., Goding et al., Monoclonal Antibodies: Principles and Practice (2d ed.) Acad.
  • the antibodies generated can be used for a number of purposes, e.g., as probes in immunoassays, for inhibiting interaction between AGEs and their receptors, in diagnostic or therapeutic applications. These applications are discussed in greater detail, below. Where the antibodies are used to block the interaction between AGEs and their receptors, the antibody will generally be referred to as a "blocking antibody.”
  • the antibodies of the present invention can be used with or without modification. Frequently, the antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels include those that are well known in the art, such as the labels described previously for the polypeptides of the invention, e.g., radioactive, fluorescent or bioactive labels.
  • the antibodies of the invention may be particularly useful in, e.g., diagnostic applications, for identifying abnormal levels of RAGE in human tissue or blood samples which abnormal levels may be indicative of the existence of or enhanced potential for those disorders associated with excessive RAGE/AGE interaction, as described herein.
  • the antibodies of the present invention may be used as affinity ligands in the quantitation and/or purification of the RAGE polypeptides from a mixture of proteins.
  • affinity purification methods are well known in the art, and typically involve the immobilization of a particular antibody, e.g., an antibody to soluble human RAGE, upon a solid support.
  • Solid supports for use in affinity chromatography are generally commercially available from, e.g., Sigma Chemical Co. (St Louis MO) and Pharmacia (Uppsala, Sweden).
  • the antibodies of the invention may be chimeric, human- like or humanized, in order to reduce their potential antigenicity, without reducing their affinity for their target.
  • Chimeric, human-like and humanized antibodies have generally been described in the art.
  • such chimeric, human-like or humanized antibodies comprise hypervariable regions, e.g., complementarity determining regions (CDRs) from a mammalian animal, i.e., a mouse, and a human framework region.
  • CDRs complementarity determining regions
  • hybrid antibodies By incorporating as little foreign sequence as possible in the hybrid antibody, the antigenicity is reduced.
  • Preparation of these hybrid antibodies may be carried out by methods well known in the art.
  • Preferred antibodies are those monoclonal or polyclonal antibodies which specifically recognize and bind to human RAGE proteins and more particularly, those that specifically bind to the human soluble RAGE polypeptides of the invention. Accordingly, these preferred antibodies will specifically recognize and bind the polypeptides which have an amino acid sequence that is substantially homologous to the amino acid sequence shown in Figure 1A and 1B (Seq ID NOS: 1-4) , or immunologically active fragments thereof.
  • antibodies which are capable of forming an antibody-ligand complex with the polypeptides of the invention, whereby the ability of the RAGE polypeptides to associate with their ligands, in vitro, is reduced, e.g., blocking antibodies.
  • blocking antibodies which inhibit or reduce binding of RAGE to other natural and pathology associated ligands of human RAGE, e.g., amphoterin, ⁇ -amyloid peptides, and the like.
  • the present invention provides nucleic acids which encode the polypeptides of the invention, as well as expression vectors that include these nucleic acids, and cell lines and organisms that are capable of expressing these nucleic acids. These nucleic acids, expression vectors and cell lines may generally be used to produce the polypeptides of the invention.
  • the isolated nucleic acids of the present invention encode a polypeptide which is derived from or related to a soluble human RAGE polypeptide or biologically active fragment thereof.
  • the nucleic acid compositions of the invention will typically include a coding region which encodes a polypeptide having an amino acid sequence that is substantially homologous to the amino acid sequence shown in Figure 1A and 1B (Seq ID NOS: 1-4) .
  • Preferred nucleic acids will typically encode polypeptides having an amino acid sequence which is substantially homologous to the amino acid sequence shown in Figure 1A and 1B (Seq ID NOS: 1-4) , or biologically active fragments thereof. Such fragments will generally comprise a segment of from about 15 to about 150 nucleotides.
  • fragments can be useful as oligonucleotide probes in the methods of the present invention, or alternatively to encode the polypeptides or biologically active fragments of the present invention, described herein. Also provided are substantially similar nucleic acid sequences, allelic variations and natural or induced sequences of the above described nucleic acids. Also included are chemically modified and substituted nucleic acids, e.g., those which incorporate modified nucleotide bases or which incorporate a labelling group.
  • nucleic acids will comprise a segment having more than about 20 contiguous nucleotides from the nucleotide sequences shown in either of Figure 1A or 1B (Seq ID NOS: 1-4) , with still more preferred nucleic acids having a nucleotide sequence that is substantially homologous to either of the nucleotide sequences shown in Figure 1A or 1B (Seq ID NOS: 1-4) .
  • Most preferred nucleic acids are those which include a portion, i.e., at least 20 contiguous nucleotides, or all of the nucleotide sequence shown in Figures 1A or B (Seq ID NOS: 1-4) .
  • Nucleic acids of the present invention include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands. Furthermore, different alleles of each isoform are also included.
  • the present invention also provides recombinant nucleic acids which are not otherwise naturally occurring.
  • the nucleic acids described herein also include self replicating plasmids and infectious polymers of DNA or RNA. Unless specified otherwise, conventional notation for nucleic acids is used herein. For example, as written, the left hand end of a single stranded polynucleotide sequence is the 5'-end, whereas the right-hand end is the 3'-end.
  • the left hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • nucleic acid sequence encoding refers to a nucleic acid which directs the expression of a specific protein or peptide.
  • the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
  • the nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length protein. It will be further understood that the nucleic acids of the invention also encompass degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • Substantial homology in the nucleic acid context means that the segments, or their complementary strands, when compared, are the same when properly aligned, with the appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, typically, at least about 70%, more typically, at least about 80%, usually, at least about 90%, and more usually, at least about 95% to 98% of the nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions to a strand, or its complement, typically using a sequence of at least about 20 contiguous nucleotides derived from the nucleotide sequences shown in Figures 1A or 1B (Seq ID NOS: 1- 4) .
  • larger segments will usually be preferred, e.g., at least about 30 contiguous nucleotides, more usually about 40 contiguous nucleotides, and preferably more than about 50 contiguous nucleotides.
  • Selective hybridization exists when hybridization occurs which is more selective than total lack of specificity. See, Kanehisa, Nucleic Acid Res. 12:203-213 (1984). Examples of such selective hybridization conditions include, e.g., hybridization under the hybridization and wash conditions of 50% formamide at 42°C. Other stringent hybridization conditions may also be selected. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C.
  • the combination of parameters is more important than the absolute measure of any one.
  • nucleic acids of the present invention may be present in whole cells, cell lysates or in partially pure or substantially pure or isolated form.
  • substantially pure or isolated generally refer to the nucleic acid separated from contaminants with which it is generally associated, e.g., lipids, proteins and other nucleic acids.
  • the substantially pure or isolated nucleic acids of the present invention will be greater than about 50% pure. Typically, these nucleic acids will be more than about 60% pure, more typically, from about 75% to about 90% pure and preferably from about 95% to about 98% pure.
  • the DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes specific for sequences in the desired DNA. Restriction endonuclease digestion of genomic DNA or cDNA containing the appropriate genes can be used to isolate the DNA encoding the polypeptides of the invention. From the nucleotide sequence given in Figures 1A or 1B (Seq ID NOS: 1-4) , a panel of restriction endonucleases can be constructed to give cleavage of the DNA in desired regions, i.e., to obtain segments which encode biologically active polypeptides or fragments of the invention.
  • DNA encoding the polypeptides of the invention is identified by its ability to hybridize with a nucleic acid probe in, for example, a Southern blot format. These regions are then isolated using standard methods. See, e.g., Sambrook, et al., supra.
  • PCR polymerase chain reaction
  • PCR technology is used to amplify nucleic acid sequences of the desired nucleic acid, e.g., the DNA which encodes the polypeptides of the invention, directly from mRNA, cDNA, or genomic or cDNA libraries.
  • solid phase oligonucleotide synthesis methods may also be employed to produce the nucleic acids described herein. Such methods include the phosphoramidite method described by, e.g., Beaucage and Carruthers, Tetrahedron Lett. 22:1859-1862 (1981), or the triester method according to Matteucci, et al., J.
  • a double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Appropriate primers and probes for amplifying the nucleic acids described herein may be generated from analysis of the nucleic acid sequences described herein, e.g., in Figure 1A or 1B (Seq ID NOS: 1-4) . Briefly, oligonucleotide primers complementary to the two 3' borders of the DNA region to be amplified are synthesized. The PCR is then carried out using the two primers. See, e.g., PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990).
  • Primers can be selected to amplify a variety of different sized segments from the nucleic acid sequence.
  • the nucleic acid sequences described herein are also particularly useful in a number of other applications.
  • the nucleic acid sequences of the present invention or fragments thereof may be readily employed as nucleic acid probes useful in obtaining genes which encode the polypeptides of the present invention or other closely related genes.
  • "Nucleic acid probes" may be DNA or RNA fragments.
  • DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite or phosphotriester methods described in, e.g., Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press (1990). Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid.
  • Typical nucleic acid probes may be readily derived from the nucleotide sequence shown in Figure 1A or B (Seq ID NOS: 1-4), or alternatively, may be prepared from the amino acid sequence of soluble human RAGE polypeptides, as shown in Figure 1A or 1B (Seq ID NOS: 1-4).
  • probes may be prepared based upon segments of the amino acid sequence which possess relatively low levels of degeneracy, i.e., few or one possible nucleic acid sequences which encode therefor. Suitable synthetic DNA fragments may then be prepared.
  • nucleic acid probes e.g., cDNA probes
  • cDNA probes may be used in the design of oligonucleotide probes and primers for screening and cloning genes which encode the polypeptides of the invention or related polypeptides, e.g., using well known PCR techniques.
  • These nucleic acids, or fragments may comprise part or all of the cDNA sequence that encodes the polypeptides of the present invention.
  • Effective cDNA probes may comprise as few as 15 consecutive nucleotides in the cDNA sequence, but will often comprise longer segments. Further, these probes may further comprise an additional nucleotide sequence, such as a transcriptional primer sequence for cloning, or a detectable group for easy identification and location of complementary sequences.
  • probes that are particularly useful in amplifying the nucleic acid sequence encoding soluble human RAGE as shown in Figure 1A or 1B, include those having the following sequences: 5'-GATGGCAGCCGGAACAGCAGTT-3' (Seq ID NO: 22); and 5'- CTCAAGTTCCCAGCCCTGATCCTCC-3' (Seq ID NO: 23).
  • cDNA or genomic libraries of various types may be screened for new alleles encoding RAGE or related sequences, using the above probes.
  • the choice of cDNA libraries normally corresponds to tissue sources which are abundant in mRNA for the desired polypeptides, e.g., lung tissue. Phage or plasmid libraries may generally be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured, and probed for the presence of the desired sequences.
  • nucleic acids of the present invention may also comprise a segment encoding a heterologous protein, such that the gene is expressed to produce the two proteins as a fusion protein, as substantially described above.
  • nucleic acids of the present invention will be used in expression vectors for the preparation of the polypeptides of the present invention, namely those polypeptides which are derived from or related to soluble human
  • expression vector generally refers to nucleotide sequences that are capable of affecting expression of a structural gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
  • DNA encoding the RAGE polypeptides of the present invention will typically be incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Often, the nucleic acids of the present invention may be used to produce a suitable recombinant host cell. Specifically, DNA constructs will be suitable for replication in a prokaryotic host, such as bacteria, e.g., E.
  • DNA constructs prepared for introduction into a particular host will typically include a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide encoding segment.
  • a DNA segment is operably linked when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide.
  • DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase.
  • enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
  • the selection of an appropriate promoter sequence will generally depend upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art.
  • the transcriptional regulatory sequences will typically include a heterologous enhancer or promoter which is recognized by the host.
  • the selection of an appropriate promoter will depend upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available. See Sambrook et al., (1989).
  • expression vectors which include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment may be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., and in Metzger et al., Nature 334:31-36 (1988).
  • suitable expression vectors may be expressed in, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli.
  • the cDNA encoding the polypeptides of the invention may be cloned into an appropriate baculovirus expression vector, e.g., pBacPAK8 vector (Clontech, Palo Alto, CA).
  • the recombinant baculovirus may then be used to transfect a suitable insect host cell, e.g., Spodoptera frugiperda (Sf9) cells, which may then express the polypeptide.
  • a suitable insect host cell e.g., Spodoptera frugiperda (Sf9) cells, which may then express the polypeptide.
  • Sf9 Spodoptera frugiperda
  • compositions of the present invention have a wide variety of uses, including, inter alia, screening, diagnostic and therapeutic applications.
  • the polypeptides of the invention may be used as model systems for identifying effectors of the AGE/RAGE interaction.
  • these model systems may be used to screen collections or libraries of test compounds in order to identify agonists or antagonists of AGE/RAGE interaction.
  • agonists, antagonists or test compounds may be chemical compounds, mixtures of chemical compounds, biological macromolecules, or extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues.
  • Particularly targeted test compounds will typically include the polypeptides or fragments of the present invention as well as structural analogs or peptidomimetics which are derived from these polypeptides or the antibodies described herein, substrates or ligands thereof.
  • agonist refers to a composition or compound that will enhance the particular observed activity, e.g., AGE/RAGE binding, while an “antagonist” will diminish the particular observed activity.
  • agonist and antagonist do not imply any particular mechanism of function.
  • the screening methods of the present invention typically involve the incubation of a polypeptide of the present invention, e.g., a soluble human RAGE polypeptide, in the presence of a standard advanced glycosylation end-product protein (AGE) such as AGE-BSA, nonenzymatically N-glycosylated collagen, myelin or the like, as well as the test compound.
  • AGE advanced glycosylation end-product protein
  • one of the RAGE polypeptide or AGE will be immobilized upon a solid support which will then be contacted with the other protein or peptide.
  • the other, non-immobilized member of the AGE/RAGE pair will typically include a labeling group covalently or otherwise attached so as not to interfere with the AGE/RAGE interaction.
  • Labeling groups will generally include those that are substantially set out above. Immobilization of one of the AGE or RAGE polypeptide permits ready separation of AGE/RAGE complex, which will be bound to the solid support, from unreacted or free AGE or RAGE, utilizing a simple wash step.
  • suitable solid supports may be employed for immobilization of the AGE or RAGE polypeptides.
  • suitable solid supports include agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose, polystyrene, filter paper, nitrocellulose, ion exchange resins, plastic films, glass beads, polyaminemethylvinylether maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the support may be in the form of, e.g., a test tube, microtiter plate, beads, test strips, or the like.
  • the reaction of the AGE or RAGE polypeptide with the particular solid support may be carried out by methods well known in the art.
  • supports bearing lectins e.g., Con-A
  • supports bearing lectins e.g., Con-A
  • AGEs may be covalently attached
  • a variety of pre-derivatized solid supports to which AGEs may be covalently attached are generally available from, e.g., Sigma Chemical Co. (St. Louis, MO), and Pharmacia (Upsalla, Sweden).
  • the test compound may be added to the well of the microtiter plate to preincubate with the immobilized AGE or RAGE polypeptide.
  • the remaining member of the RAGE/AGE pair, bearing a suitable labeling group as described previously, may then be added to the microtiter well.
  • the wells are washed, and the amount of bound label is determined, e.g., by scanning the plate with a suitable optical reader, e.g., plate reader.
  • the level of binding is then compared to suitable positive and negative controls or a set of standards containing a known range of agonists or antagonist concentrations.
  • suitable positive and negative controls or a set of standards containing a known range of agonists or antagonist concentrations.
  • identification of complexed AGE/RAGE may be carried out by other means, i.e., without the use of a support bound peptide.
  • well known quantitation methods such as HPLC and the like may be utilized to separate and identify complexed AGE/RAGE polypeptides from the free or uncomplexed proteins. Again, this may allow determination and comparison of the amount of either the free or bound material remaining after incubation with the test compound.
  • test compounds which are indicated to be antagonists of the RAGE/AGE interaction may be further characterized in additional studies, e.g., clinical trials. 25
  • the peptides of the invention may also be used as affinity ligands which specifically bind to AGEs.
  • affinity ligands these polypeptides may also be useful in the purification of AGEs from a mixture of different proteins. Affinity purification of AGEs may be carried out using affinity purification methods that well known in the art.
  • affinity purification methods that well known in the art.
  • the soluble RAGE polypeptide or peptides may be attached to a suitable solid support as described above. Many solid supports are commercially available from, e.g., Sigma Chemical Co., St Louis, Missouri, or Pharmacia, Uppsala, Sweden, and come prepared for immediate coupling of affinity ligands.
  • the mixture of proteins may be contacted with the polypeptide bound to the solid support, such that the RAGE polypeptide immobilized upon the solid support can selectively bind the AGEs within the mixture of proteins.
  • the bound protein can then be washed to eliminate unbound proteins.
  • substantially pure AGEs may be eluted from the solid support by generally known elution protocols, e.g., changing buffer conditions, temperature, or level of carbohydrate in the elution buffer.
  • the polypeptides of the invention may also be used to bind AGEs both in vitro and in vivo. This binding may be used in assay formats to label and detect AGEs in a sample, imaging formats to identify localization of
  • AGEs in a patient or in therapeutic applications to deliver a drug to areas which are relatively high in AGE concentration, or specifically deliver a drug, e.g., a proteolytic drug, to an AGE.
  • a drug e.g., a proteolytic drug
  • the antibodies of the invention may also be used as affinity probes or ligands for soluble RAGE polypeptides.
  • affinity probes or ligands for soluble RAGE polypeptides may be exploited in the purification and/or identification of RAGE polypeptides and particularly, soluble human RAGE polypeptides.
  • the polypeptides of the invention may be used as probes capable of specifically interacting with their ligands, i.e., AGEs.
  • the polypeptides of the invention may be used in a variety of diagnostic applications.
  • those polypeptides of the invention that are capable of specifically interacting with AGEs may be particularly useful in identifying patients who may suffer from abnormal levels of AGEs which are indicative of particular disorders, or may be viewed as indicators of future problems, such as diabetic vasculopathy.
  • soluble human RAGE polypeptides may be used as affinity probes to identify the presence, absence and/or relative quantity of AGEs in a sample, e.g., blood or tissue samples from a patient.
  • AGE or other ligands of RAGE which levels are indicative of a variety of pathological conditions associated with, e.g., Diabetes Mellitus, peripheral occlusive vascular diseases, hemodialysis- associated amyloidosis, Alzheimer's disease and other age-related disorders.
  • appropriate preventative or therapeutic measures may be taken, such as administration of appropriate pharmaceutical agents, e.g., compositions comprising the peptides, peptidomimetics or antibodies of the invention.
  • the antibodies of the present invention may be used to diagnose disorders characterized by abnormal levels or localization of
  • the described antibodies may be used as diagnostic tools to evaluate plasma and tissue levels of RAGE in patients suffering from pathological conditions associated with elevated levels of AGE/RAGE interaction.
  • the antibodies described herein may be used in well known immunoassay formats, e.g., ELISA, Western blotting, immunohistochemistry and FACS methods, to identify levels of RAGE in samples.
  • the soluble RAGE polypeptides and antibodies of the invention may also be used in therapeutic applications for the treatment of human or non-human mammalian patients.
  • treatment refers to the full spectrum of treatments for a given disorder from which the patient is suffering, including alleviation of one, most or all symptoms resulting from that disorder, to an outright cure for the particular disorder or prevention of the onset of the disorder.
  • the polypeptides and antibodies of the invention are useful in treating disorders or symptoms of which result from excessive levels of AGEs in tissue or plasma.
  • association of AGEs and RAGE has been implicated as a symptom or causative event in a number of pathological conditions including, e.g., complications associated with Diabetes Mellitus, e.g., diabetic microvasculopathy (neuropathy, nephropathy and retinopathy), diabetic macrovasculopathy (atherosclerosis), occlusive vascular disorders, activation of microglial cells by ⁇ -amyloid peptides in Alzheimer's disease, hemodialysis-associated amyloidosis and age related disorders such as oxidant stress.
  • treatment or prevention of such disorders may generally be carried out by reducing, inhibiting or outright blocking the interaction between AGEs and RAGE.
  • Blocking this interaction typically involves administering to a patient an effective amount of a soluble human RAGE polypeptide, peptidomimetc or blocking antibody, as described above.
  • patient generally refers to a mammalian individual, typically human, who has been diagnosed as suffering from one or more of the above described disorders, or who has been characterized as belonging to a group that has an abnormally high incidence of such disorders, e.g., diabetics and the elderly.
  • effective amount or “therapeutically effective amount” generally refers to the quantities of reagents necessary for effective therapy, i.e., the partial or complete alleviation of the symptom or disorder for which treatment was sought. Included within the definition of effective therapy are preventative treatments intended to reduce the likelihood of onset of the above-described symptoms or disorders.
  • the effective amount for a given therapy will generally depend upon many different factors, including means of administration, target site, physiological state of the patient and other medicants administered. Thus, treatment doses will need to be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Generally, therapeutically effective amounts of the polypeptides or blocking antibodies of the present invention will be from about 0.0001 to about 10 mg/kg, and more usually, from about 0.001 to about 0.1 mg/kg of the host's body weight.
  • Suitable pharmaceutically acceptable carriers include water, saline, buffers and other compounds described in, e.g., the Merck Index, Merck and Co., Rahway, New Jersey.
  • a liposomal formulation For some methods of administration, e.g., oral, it may be desirable to provide the active ingredient in a liposomal formulation. This is particularly desirable where the active ingredient may be subject to degradative environments, for example, proteolytic digestive enzymes.
  • Liposomal formulations are well known in the art, and are discussed in, e.g., Remington's Pharmaceutical Sciences, supra. Administration may also be carried out by way of a controlled release composition or device, whereby a slow release of the active ingredient allows continuous administration over a longer period of time.
  • Constituents of pharmaceutical compositions include those generally known in the art for the various administration methods used.
  • oral forms generally include powders, tablets, pills, capsules, lozenges and liquids.
  • intravenous, intraperitoneal or intramuscular formulations will generally be dissolved or suspended in a pharmaceutically acceptable carrier, e.g., water, buffered water, saline and the like.
  • these compositions may include additional constituents which may be required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like.
  • nontoxic solid carriers may be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like.
  • Administration may also be carried out by way of a controlled release composition or device, whereby a slow release of the active ingredient allows continuous administration over a longer period of time.
  • a DNA fragment coding for human soluble RAGE was obtained from lung cDNA library using polymerase chain reaction techniques (PCR, GeneAmp, Perlin-Elmer Cetus); primers used were 5'-GATGGCAGCCGG AACAGCAGTT-3' (Seq ID NO: 22) and 5'-CTCAAGTTCCCAGCCCTGATCCTCC-3' (Seq ID NO: 23).
  • PCR polymerase chain reaction techniques
  • primers used were 5'-GATGGCAGCCGG AACAGCAGTT-3' (Seq ID NO: 22) and 5'-CTCAAGTTCCCAGCCCTGATCCTCC-3' (Seq ID NO: 23).
  • the DNA sequence of the PCR product was confirmed by the dideoxy chain termination method (Sanger, et al., Proc. Nat'l Acad. Sci. USA 74:5463-5467
  • the DNA fragment was subcloned into the pCRIITM vector (Invitrogen, San Diego, CA) and the EcoRI fragment of the resulting plasmid was cloned into the pBacPAK8 vector (Clontech, Palo Alto, CA) under control of the AcMNPV polyhedrin promoter (ATCC Accession Nos. VR-2538 and VF25-39).
  • Baculovirus expression of recombinant human soluble RAGE was performed by co-transfecting the plasmid pBacPAK8/RAGE with a linearized BacPAK ⁇ viral cDNA (Clontech) into Spodoptera frugiperda (Sf9) cells according to the manufacturer's instructions.
  • Sf9 cells expressing soluble RAGE were grown as follows: Non ⁇ infected Sf 9 cells were grown in shake flasks at 28°C, to a density of 1 - 1.2 x 10 6 /ml in TNMF (Grace's with supplements from Sigma) plus 10% FBS (e.g. Hyclone) and 0.1% pluronic F-68 (Sigma), and a viability of >97%.
  • FBS e.g. Hyclone
  • pluronic F-68 pluronic F-68
  • Recombinant human soluble RAGE was purified from the Sf9 media by chromatography on an SP Sepharose fast flow column (Pharmacia) followed by a size exclusion chromatography step.
  • a 1/10 volume of 1.0 M Tris- HCl, pH 8.0 was added to Sf 9 cell media to precipitate viral proteins and the media was allowed to sit at 4°C for several hours to allow precipitation.
  • the precipitated media was centrifuged at ⁇ 3000 ⁇ m for 10 to 15 min to remove any precipitate.
  • the supernatant was diluted 1:4 with deionized water, adjusted to pH 7.5 and sterile filtered (0.2 ⁇ m).
  • the filtered supernatant was loaded onto SP Sepharose fast flow column (#17-0729-01 , Pharmacia), that had been equilibrated with 20 mM NaPO4, pH 7.5.
  • the column was eluted in a salt gradient from 0 to 0.5 M NaCl, and the fractions were analyzed by SDS-PAGE. Fractions containing RAGE were pooled, concentrated and diafiltered into PBS buffer. Further purification was obtained by applying the pooled fractions to a Superdex 200PG column, and again, fractions were analyzed by SDS-PAGE and pooled as appropriate.
  • Bovine serum albumin (from Sigma, Cat #A7888) was incubated at a concentration of 25 mg/ml in phosphate buffered saline (calcium- and magnesium- free) with 250 mM ribose, in the presence of 1.5 mM PMSF (phenylmethylsulfonyl fluoride) and 1 mM EDTA (ethylenediaminetetraacetic acid). The pH of the solution was adjusted to between 6.8 and 7.0. The solution was sterilized by filtering through 0.22 micron filters and incubated in dark, at 37°C for 6 to 8 weeks. The solution containing AGE-BSA was dialysed against calcium- and magnesium-free PBS, and stored frozen in aliquots at -20°C.
  • PMSF phenylmethylsulfonyl fluoride
  • EDTA ethylenediaminetetraacetic acid
  • a 96-well plate (Immunolon 4 from Dynatech Lab) was coated with anti-flag peptide antibodies (Eastman Kodak), 100 ⁇ l/well of 17 ⁇ l/ml in sodium bicarbonate buffer, pH 9.6) at 4°C overnight. The wells were washed and blocked as above. The wells were then incubated with Sf 9 media containing human recombinant soluble RAGE/flag fusion protein (75 ⁇ l of 1 :4 dilution with PBS) 1 to 2 h at 37°C (flag peptide: DYKDDDDK).
  • the wells were again washed as before and incubated with various concentrations of 12S I-AGE-BSA alone or in the presence of excess cold AGE-BSA (i.e., nonradioactive) in PBS containing 0.2% BSA (45 ⁇ l/well) at room temperature for 2-3 hours.
  • the sample having an excess of cold AGE-BSA was used to account for nonspecific interactions.
  • the wells were washed twice with 0.2% BSA in PBS.
  • the bound ligand was then eluted with PBS containing 1 mg/ml of heparin and 1 mg/ml of BSA (100 ⁇ l/well) by incubating at 37°C for 5 minutes and counted.
  • Figure 2 shows a graph of AGE binding to indirectly immobilized RAGE (expressed as CPM) as a function of increasing ligand concentration, in the absence and presence of excess nonradioactive ligand (Figure 2A), and corrected for nonspecific interactions (Figure 2B).
  • Binding assays were done in quadruplicate. Lactoferrin has been previously shown to bind AGEs. Li et al. Nature med. 1(10): 1057-1061 (1995). Furthermore, lactoferrin was also shown to bind to RAGE Schmidt et al., J. Biol. Chem. 269:9882-9888 (1994), Yan et al., J. Biol. Chem. 269:9889-9897 (1994). However, as is apparent from the Table 1 , binding of lactoferrin to RAGE or AGE does not facilitate further binding of AGE-BSA to RAGE.
  • Figure 3 is dose response curve for the formation of RAGE/AGE binding complex in the presence of increasing concentrations of soluble RAGE polypeptide. As shown, increasing concentration of soluble RAGE increased the levels of AGE/RAGE binding until a plateau was reached.
  • Monoclonal antibodies were generated from mice immunized with the human soluble RAGE extracellular domain expressed in baculovirus as described above.
  • Hybridoma preparation Pairs of mice from three strains (Balb/C, Swiss Webster, and RBF/DnJ) were immunized with 100 ⁇ g soluble RAGE in complete Hunter's adjuvant, intradermally, on days 0, 7, and 21. Sera were drawn on day 28, and titers tested by EIA of soluble RAGE and FACS analysis of CHO- RAGE transfectants. Two mice were selected as lymphocyte donors, and received 5 ⁇ g soluble RAGE IV 72 hours before fusion. Splenocytes from these mice were fused with the mouse myeloma P3X63Ag8.653, and the resultant fusion products were selected with hypoxanthine-aminopterin-thymidine (HAT).
  • HAT hypoxanthine-aminopterin-thymidine
  • the resultant panel was tested for reactivity by EIA, Western blot, and FACS analysis of CHO cells transfected with full length RAGE.
  • the antibodies were also analyzed for epitope variance by competition, and reactivity with RAGE/DCC chimeric protein. Complementary pairs of antibodies were selected, and antigen capture EIAs specific for human, rat, and mouse RAGE were designed, with sensitivity in the nanogram range.
  • EIA Direct antibody capture EIA was done by coating 96-well microtiter plates with 1 ⁇ g/well antigen in PBS, and incubating overnight at 4°C. Wells were blocked with PBS/1 % BSA or casein, anti-RAGE antibodies added as either neat tissue culture supernatant or 1 ⁇ g/well diluted in PBS/1% BSA and incubated for 2 hours at ambient room temperature. After washing 3X with PBS, anti-mouse IgG-alkaline phosphatase was added, and substrate degradation analyzed at 405 nm with a UVMax plate reader. Antigen capture (sandwich) EIAs were done as above, with the additional step of coating wells with 2 ⁇ g anti-RAGE monoclonal antibody diluted in carbonate buffer, pH 9.6, before addition of antigen.
  • FACS analysis Cells (CHO-RAGE, parental CHO, mouse macrophage, and human smooth muscle aorta) were harvested, and incubated with
  • the direct antibody capture EIA was used as the initial fusion screen to identify positive clones.
  • the assay format was also utilized to determine reactivity with the RAGE chimeric RAGE/DCC protein in which the first immunoglobulin-like domain was replaced with that of DCC.
  • the assay results for the first 20 MAbs are shown in Figure 4. Four MAbs recognized both intact soluble
  • RAGE polypeptide as well as the chimeric protein indicating epitope location outside the first Ig-like domain.
  • FIG. 4B shows an antigen capture assay utilizing peptide fragment # 2
  • the sandwich EIA using MAbs RBF9D9 (ATCC Accession No. HB- 12165) as capture and SW10C1-biotin (ATCC Accession No. HB-12164) as detection is specific for human RAGE, and does not cross react with either rat or mouse RAGE.
  • This assay was used to detect and quantitate the presence of RAGE expressed in baculovirus, as well as CHO cells ( Figure 6A). Controls included a concentration range of purified RAGE for generation of a standard curve, as well as RAGE-spiked sera controls; the range of linearity is 20 - 100 ng/well.
  • a second sandwich EIA, specific for human, rat, and mouse RAGE uses MAbs RBF9D9 (ATCC Accession No.
  • Bovine aortic endothelial cells (BAEC) were cult-Cured to confluency on nucleopore membranes. Seven days after reaching confluency, cells were incubated for 24 hours with red blood cells (RBCs) isolated from either normal subjects or diabetic patients. Endothelial cells were washed and permeability was measured in a permeability chamber containing minimal essential medium containing 10% fetal calf serum by adding 125 l-albumin or 3 H-inulin to the upper chamber. The emergence of radioactivity in the lower chamber was then measured over 24 hours at 37°C.
  • BAEC Bovine aortic endothelial cells
  • P permeability coefficient
  • J the flux of molecules across the filter
  • A the surface area of the confluent layer of endothelial cells
  • C the concentration of tracer in the upper chamber
  • C b the concentration of tracer in the lower chamber.
  • Post-confluent monolayers displaying permeability coefficients greater than 6.5 X 10 "7 cm/s , for albumin, or greater than 5 X 10 '6 cm/s for inulin were excluded.
  • RBCs were collected from normal or diabetic rats by puncturing the lower abdominal aorta.
  • the RBCs were collected in a solution of dextrose (2.4%), citric acid (2.4%), sodium citrate (0.73%) and 2 parts anticoagulant to 8 parts blood. Blood was centrifuged to remove plasma and buffy coat, and the packed RBCs were washed and infused (4.2 X 10 9 cells/animal) into normal syngeneic animals (vol. 0.5 ml). After one hour, TBIR was determined by infusion of 125 l-albumin followed 30 minutes later by infusion of 51 Cr-labelled normal RBCs. Tissue and blood samples were collected 5 minutes later.
  • Radioactivity was measured as the trichloroacetic plasma precipitable fraction.
  • Plasma human recombinant soluble RAGE concentration data were fit to a two compartment open model using nonlinear regression by extended least squares analysis (Siphar, SIMED, Cretail, France). The elimination and distribution half-lives were 26 and 0.13 hours, respectively.
  • 10 control diabetic rats were used, 7 diabetic rats were treated with soluble RAGE and 5 rats were treated with the control protein.
  • TBIR was calculated as a ratio of [ 125 l]/[ 51 Cr] in tissue over the same ratio in blood.
  • One way analysis of variance followed by Dunnet's test was used to analyze the data for each organ. The results for normal and diabetic rats are given in figures 12A and 12B, respectively.
  • TBIR increased in a number of tissues as compared to infusion of normal RBCs ( Figure 11 A). Most of the increases in TBIR were prevented by pretreatment of diabetic rats with recombinant soluble RAGE, but not with the control protein, with the exception of kidney tissue, which showed a similar effect with both RAGE and VCAM-1. These results indicate that AGEs on the surface of diabetic RBCs interact with surface RAGE on endothelial cells, triggering activation of the latter cells. This leads to an increased permeability of the endothelial layers. Without being bound to a particular theory, it is believed that pretreatment of diabetic RBCs with soluble RAGE prevents this interaction and, as a result, prevents increases in permeability as demonstrated.
  • TBIR also increased in various tissues of diabetic rats as compared to those of normal rats.
  • recombinant soluble RAGE In other tissues, e.g., intestine, skin, both recombinant soluble RAGE and control protein had some effects (Figure 11B).
  • TBIR results in diabetic rats were not as clear as those for normal rats. This is believed to be a result of diabetes associated changes in hemodynamic factors that may effect TBIR. Accordingly, additional assays were performed to confirm the efficacy of RAGE in preventing diabetes associated increases in permeability.
  • Recombinant soluble RAGE purified according to the methods described above was administered at infusion rates to achieve plasma concentrations of 60-80 ⁇ g/ml for 1.5 hours. Arterial pressure, right atrial pressure, and body temperature were continuously monitored. Western blotting was performed on plasma samples and standards to determine the level of circulating recombinant protein during the studies.
  • tissue samples were surveyed in the studies: skin (hindlimb and back); skeletal muscle (gastrocnemius, tibialis anterior, abdominal wall); heart (left ventricle); lung (right and lefty lower lobe); lower trachea; aorta, sciatic nerve; retina; kidney; pancreas, jejunum, ileum, colon, testis, cerebrum and visceral fat pad.
  • Initial and final plasma volumes were estimated as the 5 minutes 131 I-RSA and 125 I-RSA distribution volumes, respectively.
  • Tissue extravascular water contents (EVW) were determiend as (wet weight)-( 125 l-RSA volume)-(dry weight). Values of C RSA and EVW were normalized to tissue blood-free dry weight. The data were separated into early (2-3 weeks), mid (4-10 weeks), and late (11-20 weeks) phases following streptozocin injection for comparison.
  • MOLECULE TYPE DNA (genomic)
  • GTAGGTGCTC AAAACATCAC AGCCCGGATT GGCGAGCCAC TGGTGCTGAA GTGTAAGG 120
  • CCCCCCAGCC CTGTGCTGAT CCTCCCTGAG ATAGGGCCTC AGGACCAGGG AACCTACAGC 900 TGTGTGGCCA CCCATTCCAG CCACGGGCCC CAGGAAAGCC GTGCTGTCAG CATCAGCATC 960
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:23:

Abstract

La présente invention a été notamment dirigée sur des compositions réagissant de manière spécifique avec des produits terminaux d'une glycosylation avancée (AGE) ou avec les récepteurs de ceux-ci. On peut utiliser de telles compositions dans de nombreuses applications, notamment dans des applications thérapeutiques, par exemple en tant qu'agents de blocage pour inhiber ou diminuer d'une autre façon l'interaction AGE/RAGE, dans des applications de criblage, par exemple comme modèles de l'interaction AGE/RAGE, et dans des applications de diagnostic, par exemple pour identifier des taux anormaux d'AGE ou de RAGE dans un système donné.
PCT/EP1997/001832 1996-04-16 1997-04-11 Peptides du recepteur de produits terminaux d'une glycosylation avancee, et utilisation de ces peptides WO1997039121A1 (fr)

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WO2001012598A2 (fr) * 1999-08-13 2001-02-22 The Trustees Of Columbia University In The City Of New York Procedes d'inhibition de la liaison de la fibrille a feuillets beta au recepteur rage, et leurs consequences
WO2001092892A2 (fr) * 2000-05-30 2001-12-06 Transtech Pharma, Inc. Procedes d'identification de composes qui modulent le recepteur rage
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WO2005051995A2 (fr) * 2003-11-19 2005-06-09 Curagen Corporation Nouvelle proteine de type recepteur specifique au produit d'extremite d'une glycosylation avancee et acides nucleiques codant cette proteine
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