WO2008137552A2 - Anti-rage antibodies and methods of use thereof - Google Patents

Anti-rage antibodies and methods of use thereof Download PDF

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Publication number
WO2008137552A2
WO2008137552A2 PCT/US2008/062231 US2008062231W WO2008137552A2 WO 2008137552 A2 WO2008137552 A2 WO 2008137552A2 US 2008062231 W US2008062231 W US 2008062231W WO 2008137552 A2 WO2008137552 A2 WO 2008137552A2
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Prior art keywords
antibody
antibodies
rage
seq
variable region
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PCT/US2008/062231
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French (fr)
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WO2008137552A3 (en
Inventor
Su-Yau Mao
Bo Chen
Chew-Shun Chang
Jane Tian
Partha Chowdhury
Anthony Coyle
Gary Sims
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Medimmune, Llc
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Publication of WO2008137552A3 publication Critical patent/WO2008137552A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • RAGE receptor for advanced glycation end products
  • RAGE ligands include advanced glycation end products (AGE), amyloid- ⁇ (A- beta; A ⁇ ) peptide, proinflammatory cytokine-like mediators of the SlOO/calgranulin family, and high mobility group box 1 protein (HMGBl).
  • AGE advanced glycation end products
  • A- beta amyloid- ⁇
  • HMGBl high mobility group box 1 protein
  • RAGE has been implicated in several inflammatory disorders and diabetes, and recently we reported that HMGBl and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis (see, e.g., Tian et al., 2007, Nat.
  • IFN ⁇ levels are associated with systemic lupus erythematosus (SLE) and are believed to play an important role in disease pathology.
  • SLE systemic lupus erythematosus
  • RAGE has also been shown to have an active, pathogenic role in a wide range of amyloidogenic diseases and disorders, including Alzheimer's disease.
  • a number of significant human disorders are associated with an increased production of ligands for RAGE or with increased production of RAGE itself. Consistently effective therapeutics are not available for many of these disorders. These disorders include, for example, many chronic inflammatory diseases, including rheumatoid and psoriatic arthritis, SLE, chronic obstructive pulmonary disease (COPD), scleroderma, and intestinal bowel disease, cancers, diabetes and diabetic nephropathy, amyloidoses, Alzheimer's disease, cardiovascular diseases, and sepsis. It would be beneficial to have safe and effective treatments for such RAGE-related diseases and disorders.
  • COPD chronic obstructive pulmonary disease
  • the invention is based in part on the discovery of antibodies that bind RAGE and inhibit the binding between RAGE and HMGBl complexed with CpG DNA.
  • the anti- RAGE antibodies of the present invention also referred to herein as "antibodies of the invention” were also seen to inhibit type I IFN-inducible gene expression associated with systemic lupus erythematosus (SLE).
  • the present invention provides methods of inhibiting the interaction of RAGE and HMGBl and/or HMGB 1/CpG complexes with RAGE by administering the anti-RAGE antibodies of the instant invention.
  • the present invention also provides methods of inhibiting type I IFN-inducible gene expression associated with SLE.
  • the invention also includes methods for preventing or treating a RAGE- related disease or disorder in a subject having such a disease or disorder, that comprises administering to the subject a therapeutically effective amount of an anti-RAGE antibody or a RAGE-binding fragment thereof of the invention.
  • the present invention provides methods of treating a subject having a disease or disorder characterized by amyloid deposit of A ⁇ by administering a therapeutically effective amount of an antibody that binds specifically to RAGE (i.e., anti-RAGE antibodies) and inhibits the binding of a RAGE ligand (e.g., HMGB, A ⁇ ), also referred to herein as a "binding partner".
  • RAGE i.e., anti-RAGE antibodies
  • a RAGE ligand e.g., HMGB, A ⁇
  • the diseases or disorders treatable by the disclosed methods may be characterized by amyloid deposit of A ⁇ in brain, such as occurs in Alzheimer's disease.
  • Anti- RAGE antibodies as described herein may also be used to inhibit or reduce accumulation of amyloid deposit of A ⁇ in a subject, to inhibit or reduce neurodegeneration in a subject, to inhibit or reduce cognitive decline in a subject, and/or to improve cognition in a subject.
  • the invention includes a method for preventing or treating a RAGE-related disease or disorder selected from the group consisting of sepsis, septic shock, including disorders or conditions such as community-acquired pneumonia, which result in sepsis or septic shock, listeriosis, inflammatory diseases, scleroderma, cancers, arthritis, rheumatoid arthritis, peritonitis, Crohn's disease, lupus, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury (SCI), allograft rejection, chronic acute inflammatory diseases, cardiovascular diseases, erectile dysfunction, diabetes, complications of diabetes, vasculitis, nephropathies, retinopathies, neuropathies, amyloidoses, preclinical Alzheimer's disease, Alzheimer's disease, and CO
  • Such a method of the invention can comprise administering a composition comprising an anti-RAGE antibody or RAGE-binding fragment thereof of the invention in combination with one or more agents useful in the treatment of the RAGE-related disease or disorder that is to be treated.
  • agents of the invention include antibiotics, anti-inflammatory agents, antioxidants, ⁇ -blockers, antiplatelet agents, ACE inhibitors, lipid-lowering agents, anti-angiogenic agents, and chemotherapeutics.
  • the antibodies of the invention are useful for diagnostic applications.
  • FIG. 1 Alignment of the amino acid sequences of human, bovine (Neeper et ah, 1992), and rat (Renard et al, 1997) RAGE. Potential N-glycosylation sites are shown by bold underlining, and tyrosine residues (Y) are shown by light underlining. Cysteine residues involved in Ig domains are marked by asterisks. Boxed amino acids correspond to residues different among the three RAGE molecules.
  • FIG. 1 Schematic diagram of human RAGE (huRAGE) depicting the domains and structure of RAGE and the various huRAGE deletion constructs used in the studies described in the Examples.
  • Figure 3 Example of reactive epitope mapping of anti-huRAGE antibody clone SNSR2.62#5 showing binding of this antibody clone to the Cl region.
  • Figure 4 Example of Inhibition of HMGB-1/CpG binding to immobilized huRAGE-Fc by N7 (SNSR7#2) and N262 (SNSR2.62#5).
  • An unrelated mouse IgG antibody was used as a control.
  • FIG. 1 Profiles of the effect of anti-huRAGE mAbs on the relative gene expression of IFITl, G1P2 (Panel A, top and bottom, respectively), MXl and RSAD2 (Panel B, top and bottom, respectively) in human PBMCs stimulated with sera from SLE patients.
  • Figure 6 shows bar graphs depicting the relative expression of several IFN- ⁇ inducible genes (top row from left to right: IFITl, MXl, and DDX58; bottom row from left to right G1P2, OAS3, and RSAD2) by healthy human donor PBMCs following stimulation by 50% sera from SLE patients alone ("SLE") or in the presence of either the anti-RAGE Ab LpxR30#12 (“SLE / L30") or an unrelated control antibody (“SLE / Cont IgG”).
  • Figure 7 The amino acid sequences and nucleotides encoding the variable regions of the light chains (V L ) and the heavy (V H ) of the mouse anti-RAGE antibodies of the invention.
  • CDRs Kabat definition.
  • the invention is based in part on the discovery of antibodies that bind to the Receptor for Advanced Glycation End Products (RAGE) and inhibit the binding between RAGE and HMGBl alone or complexed with CpG DNA (such complexes are also referred to herein as "HMGB 1/CpG").
  • the HMGB (High Mobility Group Box) proteins are abundant chromatin-binding proteins located mainly in the nucleus where they regulate many transcriptional events by increasing the affinity of transcription factor-DNA interactions.
  • HMGB proteins HMGBl, HMGB2 and HMGB3 that are highly conserved and consist of an A-box and a B-box with a carboxy-terminal tail.
  • HMGBl is released from cells by two distinct mechanisms; it is passively released by necrotic cells and is also actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of the molecule (See, PCT/IB2003/005718).
  • HMGB 1 has been implicated in inflammatory disorders such as sepsis and rheumatoid arthritis (see, for example International Patent Publications WO/2007/001422; WO 2007/084253 and WO 2007/076200).
  • CpG belongs to a family of molecules referred to as "PAMPS.”
  • PAMPS are molecules which have structures called pathogen-associated molecular patterns.
  • Non-limiting examples of PAMPs are, lipopolysaccharide (LPS) from the gram-negative cell wall, peptidoglycan, lipoteichoic acids from the gram-positive cell wall, flagellin, pilin, mannose rich glycans, bacterial and viral nucleic acids, N-formylmethionine found in bacterial proteins, double-stranded RNA from viruses, phosphorylcholine and other lipids common to microbial membranes and glucans such as lipoteichoic acids, glycolipids, and zymosan from fungal cell walls.
  • HMGB can form an immunostimulatory complex with PAMPs that augments PAMP signaling.
  • antibodies of the invention may inhibit the binding of RAGE to HMGBl and/or RAGE binding to HMGB 1 complexed with a PAMP (such complexes are also referred to herein as "HMGB 1/P AMP").
  • antibodies of the invention inhibit RAGE binding to HMGBl complexed CpG.
  • antibodies of the invention inhibit RAGE binding to HMGBl complexed to human DNA (such complexes are also referred to herein as "HMGB1/DNA”).
  • an antibody of the invention inhibits RAGE binding to HMGBl associated with an immune complex (such complexes are also referred to herein as "HMGB 1/IC").
  • Immune complexes include but are not limited to those comprising cellular DNA and auto-immune antibodies such as anti-DNA antibodies.
  • the antibodies of the invention inhibit RAGE binding to one or more HMGBl complexes including, but not limited to, HMGB 1/P AMP, HMGB1/DNA and HMGB1/IC complexes.
  • the antibodies of the invention inhibit RAGE binding to one or more HMGBl complexes (e.g., HMGB 1/CpG complexes) by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%.
  • RAGE is known to bind numerous ligands in addition to HMGB 1.
  • RAGE ligands include advanced glycation end products (AGEs), adducts formed by glycation and/or glycoxidation (Baynes J.W., 1991, Diabetes. 1991, 40:405-412; Ahmed K.A., 2007, J Clin Biochem Nutr.
  • calgranulin C also known as ENRAGE and S100A12
  • SlOOAl also known as ENRAGE and S100A12
  • SlOOAl also known as ENRAGE and S100A12
  • SlOOAl also known as ENRAGE and S100A12
  • SlOOAl also known as ENRAGE and S100A12
  • S100A4 SlOOAl 1
  • S100A13 also known as SlOOB
  • SlOOP amyloid- ⁇ -peptide
  • a ⁇ amyloid- ⁇ -peptide
  • leukocyte integrins e.g., Mac-1).
  • the antibodies of the invention do not inhibit the binding of one or more RAGE ligand selected from the group consisting of: AGEs, S100A12, SlOOAl, S100A4, SlOOAl 1, S100A13, SlOOB, SlOOP, amyloid- ⁇ -peptide and Mac-1.
  • the antibodies of the invention also inhibit the binding of one or more RAGE ligand selected from the group consisting of: AGEs, S100A12, SlOOAl, S100A4, SlOOAl 1, SlOOAl 3, SlOOB, SlOOP, amyloid- ⁇ -peptide and Mac-1.
  • the antibodies of the invention selectively inhibit the binding of a subset of RAGE ligands including, but not limited to AGEs, S100A12, SlOOAl, S100A4, SlOOAI l, S100A13, SlOOB, SlOOP, amyloid- ⁇ -peptide and Mac-1.
  • Non-limiting examples of such selectively would include antibodies that inhibit the binding of RAGE to HMGBl (alone or complexed) that also inhibit the binding of AGEs and/or A ⁇ but do not inhibit the binding of one or more SlOO family members, or antibodies that inhibit inhibit the binding of RAGE to HMGBl (alone or complexed) that also inhibit the binding of one or more SlOO family members but do not inhibit the binding of AGEs and/or A ⁇ .
  • RAGE HMGBl alone or complexed
  • the antibodies of the invention inhibit the binding of RAGE to HMGBl (alone or complexed) and do not inhibit the binding of RAGE to human RAGE SlOOAl 2. In another specific embodiment, the antibodies of the invention inhibit the binding of RAGE to HMGBl (alone or complexed) and do not inhibit the binding of RAGE to bovine SlOOB.
  • the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR.
  • the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%.
  • the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR selected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRI l.
  • antibodies of the invention bind to RAGE and antigenic fragments thereof.
  • antibodies of the invention bind human RAGE and antigenic fragments thereof.
  • Human RAGE is exemplified by GenBankTM accession number BC020669 (SEQ ID NO:1). As shown in Figure 1, RAGE is highly conserved in a number of species. Accordingly, the antibodies of the invention will bind epitopes conserved across species.
  • antibodies of the invention bind murine RAGE and antigenic fragments thereof.
  • Murine RAGE is exemplified by GenBankTM accession number NM 007425.
  • antibodies of the invention bind rat RAGE and antigenic fragments thereof.
  • Rat RAGE is exemplified by GenBankTM accession number NP 445788 (SEQ ID NO:2) and in Figure 1.
  • antibodies of the invention bind bovine RAGE and antigenic fragments thereof.
  • Bovine RAGE is exemplified GenBankTM accession number NM 173982 (SEQ ID NO:3) and in Figure 1. Numerous splice variants and iso forms of RAGE have been identified (see for example GenBankTM accession numbers NM OOl 136, XM OOl 163454, DQ104254 and U.S. Patent Publication 2007008740) and one of skill in the art will recognize that antibodies of the invention may bind to one or more RAGE variants and or isoforms.
  • antibodies of the invention bind to RAGE and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human.
  • the antibodies of the invention bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a RAGE polypeptide and antigenic fragments thereof, having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human RAGE polypeptide encoded by GenBankTM accession number BC020669.
  • RAGE "fragment" described herein includes a RAGE peptide or polypeptide comprising, or alternatively consisting of (or consisting essentially of) an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, at least contiguous 250 amino acid residues, at least contiguous 275 amino acid residues, at least contiguous 300
  • antibodies of the invention bind to a soluble RAGE fragment including, but not limited to, amino acids 24-336 of SEQ ID NO: 1 and fragments thereof.
  • antibodies of the invention bind to a RAGE domain including, but not limited to, the V domain (amino acids 24- 129 of SEQ ID NO : 1 ), the C 1 domain (amino acids 130-234 of SEQ ID NO:1), the C2 domain (amino acids 235-336 of SEQ ID NO:1), the VCl domains (amino acids 24-234 of SEQ ID NO:1), and the C1C2 domain (amino acids 130-336 of SEQ ID NO:1) as disclosed by Xie et al., (2007, J.
  • antibodies of the invention bind to the C2 domain (amino acids 235-336 of SEQ ID NO: 1) of RAGE as disclosed by Xie et al, supra.
  • antibodies of the invention bind to a RAGE domain including, but not limited to, the V domain (amino acids 31-106 of SEQ ID NO:1), the Cl domain (amino acids 137-214 of SEQ ID NO:1), the C2 domain (amino acids 252-308 of SEQ ID NO:1), the VCl domains (amino acids 31-214 of SEQ ID NO:1), and the C1C2 domain (amino acids 137-308 of SEQ ID NO: 1) as disclosed by Renard et al. (2007, The J. Pharm. Exp. Therap. Vol. 290:1458-1).
  • antibodies of the invention bind to the C2 domain (amino acids 252-308 of SEQ ID NO: 1) of RAGE as disclosed by Renard et al., supra.
  • antibodies of the invention bind to a polypeptide comprising a RAGE domain including, but not limited to: the V domain, wherein said polypeptide comprises amino acids 1-121 of SEQ ID NO:1; the Cl domain, wherein said polypeptide comprises amino acids 122-233 of SEQ ID NO:1; the C2 domain, wherein said polypeptide comprises amino acids 234-342 of SEQ ID NO:1; the VCl domains, wherein said polypeptide comprises amino acids 1-233 of SEQ ID NO:1; and the C1C2 domain, wherein said polypeptide comprises amino acids 122-342 of SEQ ID NO:1, as disclosed herein (see, Section 6.1).
  • antibodies of the invention bind to a polypeptide comprising the C2 domain of RAGE, wherein said polypeptide comprises
  • Additional embodiments of the present invention are directed to antibodies of the invention that bind to a RAGE polypeptide comprising (or entirely consisting of) amino acids amino acids x to y of SEQ ID NO:1, where x represents an integer from 23 to 335, and where y represents an integer from 30 to 344.
  • antibodies of the invention bind to a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human RAGE C2 domain (amino acids 235-336 of SEQ ID NO:1) and antigenic fragments thereof.
  • the antibodies of the invention bind to a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to amino acids 234-342 of SEQ ID NO:1.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A specific, non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc.
  • 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, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package.
  • ALIGN program version 2.0
  • GCG GCG sequence alignment software package
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.accelrys.com, as available on August 31, 2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.cgc.com), using a gap weight of 50 and a length weight of 3.
  • antibodies may discriminate between the same polypeptide isolated from different sources.
  • a polypeptide of similar or identical amino acid sequence isolated from different sources may be distinguished by a number of differences including but not limited to, posttranslational modifications (e.g., phosphorylation, acetylation, methylation, glycosylation, etc.), alterations in overall structure (e.g., changes in disufide bonding and/or folding) and differences in any other molecules that the polypeptide may be associated with (e.g., salts, additional subunits such as polynucleotides and/or other polypeptides).
  • posttranslational modifications e.g., phosphorylation, acetylation, methylation, glycosylation, etc.
  • alterations in overall structure e.g., changes in disufide bonding and/or folding
  • differences in any other molecules that the polypeptide may be associated with e.g., salts, additional subunits such as polynucleotides and/or other
  • antibodies of the invention inhibit the expression of one or more type I Interferon (IFN) genes or one or more type I IFN-inducible genes including but not limited to, DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35.
  • IFN Interferon
  • antibodies of the invention inhibit the expression of one or more Type I interferon genes, or one or more type I IFN-inducible genes by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 120%, by at least 140%, by at least 160%, by at least
  • antibodies of the invention inhibit the expression of one or more type I IFN genes by at least 25%. In particular embodiments, antibodies of the invention inhibit the expression of one or more type I IFN-inducible genes by at least 25%.
  • the biochemical characteristics of the antibodies of the invention include, but are not limited to, isoelectric point (pi) and melting temperature (Tm).
  • the binding characteristics of the antibodies of the invention include, but are not limited to, binding specificity, dissociation constant (Kd), epitope, ability to distinguish between various forms and/or preparations of RAGE (e.g., recombinant, native, acetylated) and ability to bind soluble and/or immobilized antigen.
  • the functional characteristics of the antibodies of the present invention include, but are not limited to, inhibition of RAGE binding to HMGB- 1/CpG, inhibition of the intracellular or extracellular localization of a RAGE molecule with a TLR, and protection in one or more RAGE-related disease or disorder (e.g., sepsis, arthritis, Alzheimer's disease).
  • RAGE-related disease or disorder e.g., sepsis, arthritis, Alzheimer's disease.
  • antibodies of the invention bind RAGE and antigenic fragments thereof with a dissociation constant or K d (k off /k on ) of less than 10 "5 M, or of less than 10 "6 M, or of less than 10 "7 M, or of less than 10 "8 M, or of less than 10 ⁇ 9 M, or of less than 10 "10 M, or of less than 10 "11 M, or of less than 10 "12 M, or of less than 10 ⁇ 13 M, or of less than 5 xlO ⁇ 13 M, or of less than 10 "14 M, less than 5xlO "14 M, or of less than 10 "15 M, or of less than 5 X 10 15 M.
  • K d dissociation constant
  • an antibody of the invention that binds RAGE and antigenic fragments thereof has a dissociation constant or IQ (k off /k on ) of between about 10 "7 M and about 10 "8 M, between about 10 "8 M and about 10 "9 M, between about 10 "9 M and about 10 "10 M, between about 10 "10 M and about 10 "11 M, between about 10 "11 M and about 10 "12 M, between about 10 "12 M and about 10 13 M, between about 10 "13 M and about 10 '14 M.
  • an antibody of the invention that binds RAGE and antigenic fragments thereof has a dissociation constant or K J (k o ff/k on ) of between 10 M and 10 M, between 10 "8 M and 10 "9 M, between 10 "9 M and 10 "10 M, between 10 "10 M and 10 "11 M, between 10 "11 M and 10 "12 M, between 10 "12 M and 10 "13 M, between 10 "13 M and 10 "14 M.
  • K D the equilibrium dissociation constant
  • KfJk 0n - also commonly referred to as kd/ka
  • binding characteristics of the antibodies of the invention can be determined by methods known to those of skill in the art, for example, by use of surface plasmon resonance measurements, and as described herein in the Examples.
  • antibodies of the invention bind RAGE and antigenic fragments thereof with a K off rate of less than 10 ⁇ 3 s "1 , less than 5x10 ⁇ 3 s “1 , less than 10 ⁇ 4 s “1 , less than 5xlO ⁇ 4 s “1 , less than 10 ⁇ 5 s “1 , less than 5xlO ⁇ 5 s “1 , less than 10 ⁇ 6 s “1 , less than 5xlO "6 s “1 , less than 1(T 7 s "1 , less than 5xlO "7 s "1 , less than 10 "8 s “1 , less than 5xlO “8 s “1 , less than 10 "9 s “1 , less than 5x10 9 s “1 , or less than 10 "10 s “1 .
  • antibodies of the invention bind RAGE and antigenic fragments thereof with a k on rate of at least 10 5 M -1 S “1 , at least 5 x 10 5 M -1 S “1 , at least 10 6 M- 1 S “1 , at least 5 x 10 6 M- 1 S “1 , at least 10 7 M- 1 S “1 , at least 5 x 10 7 M- 1 S “1 , or at least 10 8 M " V ⁇ or at least 10 9 MV.
  • antibodies of the invention bind RAGE and antigenic fragments thereof with an equilibrium dissociation constant or K D (k off /k on ) of less than 10 "5 M, or of less than 10 "6 M, or of less than 10 "7 M, or of less than 10 "8 M, or of less than 10 "9 M, or of less than 10 ⁇ 10 M, or of less than 10 "11 M, or of less than 10 ⁇ 12 M, or of less than 10 "13 M.
  • K D equilibrium dissociation constant
  • antibodies of the invention include particular antibodies (and antigen binding fragments and derivatives thereof) that specifically bind RAGE and antigenic fragments thereof.
  • S2/1.40#9 which is abbreviated herein as “S 14"; "S2/1.50#2” which is abbreviated herein as “S15”; “S2/2.2#l” which is abbreviated herein as “S22”; "S2/2.13#5" which is abbreviated herein as “S21”; “S2/3.51#5" which is abbreviated herein as “S35”; “S2/4.146#18” which is abbreviated herein as “S41”; "S2/4.17#4" which is abbreviated herein as “S2”; “S2/6.23#2” which is abbreviated herein as “S62”; “SNSR7#2” which is abbreviated herein as "N7”;
  • Antibodies having at least one, at least two, at least three, at least four, at least five, or all six of the CDRs of these antibodies are specific embodiments of the invention.
  • Isolated polynucleotides that encode these antibodies (and antigen binding fragments thereof) are also embodiments of the invention.
  • Antibodies that bind to the same epitopes as these antibodies are also embodiments of the invention, as are antibodies that compete for binding with any of the above listed antibodies.
  • the binding and functional characteristics for S 14, S 15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and FI l are listed in Table 2, 5, and 6.
  • the nucleotide and cooresponding amino acid sequences of the H and L variable domains of the mouse anti-RAGE antibodies S2, L30, and L37 are shown in FIG. 7, panels A to C, respectively.
  • the hybridoma cell lines producing S2; L4; L30 and L37 were deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110- 2209) on June 26, 2007 and assigned ATCC Deposit Nos. PTA-8510, PTA-8509, PTA-8511 and PTA-8508, respectively. Subsequent to making the deposits, sequencing analysis revealed that L4 and L30 are independent isolates of the same clone. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Since the strains referred to are being maintained under the terms of the Budapest Treaty, they will be made available to a patent office signatory to the Budapest Treaty.
  • the present invention also encompasses variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) comprising one or more amino acid residue substitutions, additions and/or deletions in the variable light (V L ) domain and/or variable heavy (V H ) domain.
  • variants of the anti-RAGE antibodies of the invention e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1
  • V L variable light
  • V H variable heavy
  • the present invention also encompasses variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) with one or more additional amino acid residue substitutions, additions and/or deletions in one or more V L CDRS and/or one or more variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) with one or more additional amino acid residue substitutions, additions and/or deletions in one or more V L CDRS and/or one or more variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21
  • VH CDRS The antibody generated by introducing substitutions, additions and/or deletions in the V H domain, V H CDRS, V L domain and/or V L CDRS of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) can be tested in vitro and in vivo, for example, for its ability to bind to a RAGE polypeptide (by, e.g., immunoassays including, but not limited to ELISA (Enzyme-Linked Immunosorbent Assay) and BIAcore), or for its ability to inhibit one or more RAGE activity.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • CDRs residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). Specifically, residues 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35
  • CDRl 50-65
  • CDR3 95-102
  • CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al., supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
  • the invention includes antibodies having at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs of an antibody disclosed herein.
  • an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has the three CDRs of the light chain variable region of any one of S2, L4, L30 and L37.
  • an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the three CDRs of the heavy chain variable region of any one of S2, L4, L30 and L37.
  • an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein both the light and the heavy chain variable regions have the three CDRs of the light and the heavy chain variable region, respectively, of any one of S2, L4, L30 and L37.
  • Another embodiment of the present invention includes the introduction of conservative amino acid substitutions in any portion of an anti-RAGE antibody of interest, described infra (see Table 2). It is well known in the art that "conservative amino acid substitution” refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide.
  • one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide.
  • Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine).
  • Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 1.
  • Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an anti-RAGE antibody of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions.
  • the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions in the relative to the original anti-RAGE antibody.
  • the derivatives of an anti-RAGE antibody of the invention have conservative amino acid substitutions (e.g., supra) are made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to a RAGE polypeptide).
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.
  • conservative amino acid substitution also refers to the use of amino acid analogs or variants.
  • Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab') fragments, disulf ⁇ de-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above (e.g., RAGE binding antibody fragments).
  • the terms "antibody” and "antibodies” as used herein specifically includes antigen binding antibody fragments.
  • the antibodies of the present invention may be monospecific, bispecific, trispecif ⁇ c or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al, J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.
  • Antibody-like and antibody-domain fusion proteins which bind RAGE are also contemplated as antibodies of the present invention.
  • An antibody-like molecule is any molecule that has been generated with a desired binding property, see, e.g., PCT Publication Nos. WO 04/044011; WO 04/058821; WO 04/003019 and WO 03/002609.
  • Antibody- domain fusion proteins may incorporate one or more antibody domains such as the Fc domain or the variable domain.
  • the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • a large number of antibody- domain molecules are known in the art including, but not limited to, diabodies (dsFv)2 (Bera et al, 1998, J. MoI. Biol. 281:475-83); minibodies (homodimers of scFv-CH3 fusion proteins) (Pessi et al, 1993, Nature 362:367-9), tetravalent di-diabody (Lu et al, 2003 J.
  • Fc domain fusions combine the Fc region of an immunoglobulin with a fusion partner which in general can be an protein, including, but not limited to, a ligand, an enzyme, the ligand portion of a receptor, an adhesion protein, or some other protein or domain.
  • An antibody which can bind an intracellular epitope is useful for binding to and disrupting/inhibiting one or more activity of intracellular RAGE (e.g., nuclear and/or cytoplasmic RAGE).
  • An intrabody comprises at least a portion of an antibody (e.g., an scFv) that is capable of specifically binding an antigen and which has been manipulated so that it can be expressed intracellularly.
  • an intrabody does not contain sequences coding for its secretion.
  • Such antibodies will bind antigen intracellularly.
  • intrabodies of the invention retain at least about 75% of the binding effectiveness of the complete antibody (i.e., having the entire constant domain as well as the variable regions) to the antigen. In one embodiment, the intrabody retains at least 85%, at least 90%, or at least 95% of the binding effectiveness of the complete antibody.
  • Intrabodies can be localized, for example, to the following intracellular locations: endoplasmic reticulum (Munro et al, 1987, Cell 48:899- 907; Hangejorden et al, 1991, J. Biol. Chem. 266:6015); nucleus (Lanford et al, 1986, Cell 46:575; Stanton et ⁇ /., 1986, PNAS 83:1772; Harlow et al, 1985, MoI. Cell Biol. 5:1605; Pap et al, 2002, Exp. Cell Res.
  • nucleolar region (Seomi et al, 1990, J. Virology 64:1803; Kubota et al, 1989, Biochem. Biophys. Res. Comm. 162:963; Siomi et al, 1998,
  • Recombinantly expressed intrabody may be administered to a patient to mediate a prophylactic or therapeutic effect.
  • the intrabody polypeptide is associated with a "membrane permeable sequence".
  • Membrane permeable sequences are polypeptides capable of penetrating through the cell membrane from outside of the cell to the interior of the cell. When linked to another polypeptide, membrane I permeable sequences can also direct the translocation of that polypeptide across the cell membrane as well.
  • Useful membrane permeable sequence include the hydrophobic; region of a signal peptide (see, e.g., Hawiger, 1999, Curr. Opin. Chem. Biol. 3:89-94;
  • the sequence of a membrane permeable sequence can be based on the hydrophobic region of any signal peptide.
  • the signal peptides can be selected, e.g., from the SIGPEP database (see e.g., von; Heijne, 1987, Prot. Seq. Data Anal. 1:41- 2; von Heijne and Abrahmsen, 1989, FEBS Lett.; 224:439-46).
  • the membrane permeable sequence is preferably based on a signal peptide endogenous to that cell type.
  • the membrane permeable sequence is a viral protein ⁇ e.g., Herpes Virus Protein VP22) or fragment thereof (see e.g., Phelan et al, 1998, Nat. Biotechnol. 16:440-3).
  • a membrane permeable sequence with the appropriate properties for a particular intrabody and/or a particular target cell type can be determined empirically by assessing the ability of each membrane permeable sequence to direct the translocation of the intrabody across the cell membrane.
  • Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e., bispecif ⁇ c antibodies, BsAbs), antibodies with additional specificities such as trispecif ⁇ c antibodies are encompassed by the instant invention.
  • BsAbs include without limitation those with one arm directed against a RAGE epitope and the other arm directed against any other antigen.
  • Methods for making bispecif ⁇ c antibodies are known in the art. Traditional production of full-length bispecif ⁇ c antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature, 305:537-539).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm (e.g., a RAGE epitope such as the C2 domain), and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690.
  • a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • Antibodies with more than two valencies incorporating at least one hinge modification of the invention are contemplated.
  • trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
  • oligoclonal antibodies refers to a predetermined mixture of distinct monoclonal antibodies. Methods for generating oligoclonal antibodies are known in the art. See, e.g., “Examples Section", example 1, PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In certain embodiments, oligoclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell.
  • oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., RAGE). Those skilled in the art will know or can determine what type of antibody or mixture of antibodies is applicable for an intended purpose and desired need.
  • antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to a RAGE antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-RAGE antibody).
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Immunoglobulins may have both a heavy and light chain.
  • An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
  • the antibodies of the invention also encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • the terms "antibody” and “antibodies” include the antibodies which bind RAGE described herein, full length antibodies and Fc variants thereof comprising Fc regions, or fragments thereof, comprising at least one novel amino acid residue described herein fused to an immunologically active fragment of an immunoglobulin or to other proteins as described herein.
  • Such variant Fc fusions include but are not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) -Fc fusions, scFv-scFv-Fc fusions.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • Antibodies of the present invention also encompass antibodies that have half- lives (e.g., serum half-lives) in a mammal, (e.g., a human), of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.
  • half- lives e.g., serum half-lives
  • the increased half- lives of the antibodies of the present invention in a mammal results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered.
  • Antibodies having increased in vivo half- lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half- lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. 6,737056 and U.S. Patent Publication
  • the antibodies of the invention may comprise modifications/substations and/or novel amino acids within their Fc domains such as, for example, those disclosed in Ghetie et al, 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al, 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.
  • Antibodies of the invention comprising modifications/substations and/or novel amino acid residues in their Fc regions can be generated by numerous methods well known to one skilled in the art. Non- limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the variable regions of an antibody of the invention may be subcloned into a vector encoding an Fc region comprising one or modifications/substations and/or novel amino acid residues. [0064] Antibodies of the invention may also be modified to alter glycosylation, again to alter one or more functional properties of the antibody. In one embodiment, the glycosylation of the antibodies of the invention is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861.
  • an antibody of the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNAc structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • Antibodies having reduced sialyation have also been shown to have increased ADCC activity while antibodies with increased sialylation have been shown to have reduced ADCC activity and anti-inflammatory activity (Scallon et al. (2006) MoI Immunol, 7:761-72 and Kaneko et al. (2006) Science, 313:670-3).
  • Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery.
  • Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
  • the antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding a RAGE polypeptide or fragment thereof and/or generating a desired response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homo log of a polypeptide of the present invention are included. Antibodies that bind polypeptides (and polypeptide fragments) with at least 99%, at leat 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a human RAGE polypeptide (e.g., a human RAGE C2 domain) of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human RAGE proteins and the corresponding epitopes thereof.
  • a human RAGE polypeptide e.g., a human RAGE C2 domain
  • Antibodies that do not bind any other analog, ortholog or homolog or RAGE such as antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a RAGE polypeptide of the present invention are also included in the present invention.
  • antibodies or antigen binding fragments thereof that specifically bind to a RAGE polypeptide or fragment thereof prevent/antagonize/inhibit one or more of the following: RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG), expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes, including but not limited to, DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, IFI35.
  • PAMP e.g., LPS, CpG
  • Type IFN genes e.g., DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, IFI35.
  • inhibitor concentration 50% represents the concentration of an inhibitor (e.g., an antibody of the invention) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets (e.g., RAGE). It will be understood by one in the art that a lower IC50 value corresponds to a more potent inhibitor.
  • the antibodies of the invention inhibit the expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes by at least 30% with an IC50 of less than 5000 ng/ml, or of less than 4000 ng/ml, or of less than 3000 ng/ml, or of less than 2000 ng/ml, or of less than 1000 ng/ml, or of less than 500 ng/ml, or of less than 250 ng/ml, or of less than 100 ng/ml, or of less than 50 ng/ml, or of less than 10 ng/ml, or of less than 5 ng/ml.
  • the antibodies of the invention inhibit expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes by at least 30% with an IC50 of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM.
  • the antibodies of the invention inhibit RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG) with an IC50 of less than 5000 ng/ml, or of less than 4000 ng/ml, or of less than 3000 ng/ml, or of less than 2000 ng/ml, or of less than 1000 ng/ml, or of less than 500 ng/ml, or of less than 250 ng/ml, or of less than 100 ng/ml, or of less than 50 ng/ml, or of less than 10 ng/ml, or of less than 5 ng/ml.
  • a PAMP e.g., LPS, CpG
  • the antibodies of the invention inhibit RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG) with an IC50 of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM.
  • a PAMP e.g., LPS, CpG
  • EC50 effective concentration 50%
  • RAGE the maximum possible binding activity at the target molecule
  • antibodies of the invention bind to RAGE with an EC50 value of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM, or of less than 0.001 nM.
  • antibodies of the invention bind to RAGE with an EC50 value of less than 0.01 nM, or of less than 0.001 nM.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.
  • a RAGE polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et aL, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et aL, in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N. Y., 1981).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • a “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. [0078]
  • a “monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a RAGE polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et ah, J. Immunol. Methods 182:41-50 (1995); Ames et ah, J. Immunol. Methods 184:177-186 (1995); Kettleborough et ah, Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
  • Humanized antibodies are antibody molecules from non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323 (1988)).
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/technology 12:899-903 (1988)).
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • the antibody is preferably modified to make it less immunogenic in the individual.
  • the individual is human the antibody is preferably "humanized"; where the complementarity determining region(s) of the antibody is transplanted into a human antibody (for example, as described in Jones et al, Nature 321 :522-525, 1986; and Tempest et al., Biotechnology 9:266-273, 1991).
  • Phage display technology can also be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-RAGE antibodies or from naive libraries (McCafferty et al., Nature 348:552-554, 1990; and Marks, et al, Biotechnology 10:779-783, 1992).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson et al, Nature 352: 624-628, 1991).
  • polypeptides may be chosen such that the antibody generated will not significantly cross-react or specifically bind to another variant or isomer of the RAGE protein (see for example, Hudson B.I., et al., 1998, Diabetes, 47: 1155-1157; and U.S. Patent Publication 2007008740).
  • polypeptides which share a large degree of homology between two or more variants of the RAGE protein may be used for the generation of an antibody that can specifically bind ⁇ i.e., cross-react) with multiple variants or isomers of the RAGE protein.
  • an antibody of the invention will bind cross-react or specifically bind to RAGE proteins from one or more different species, including but not limited to, human, mouse, primate, and monkey.
  • Antibodies or antigen binding fragments thereof that bind to a RAGE polypeptide can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding a high affinity antibody of the invention and antigen binding fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode an antibody that specifically binds to a RAGE polypeptide (e.g., human, murine, or rat RAGE or fragments thereof).
  • a polynucleotide of the invention encodes an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:1 or fragments thereof.
  • a polynucleotide of the invention encodes an antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO: 1.
  • a polynucleotide of the invention encodes an antibody that binds to a soluble RAGE fragment including, but not limited to, amino acids 24-336 of SEQ ID NO:1 and fragments thereof.
  • a polynucleotide of the invention encodes an antibody that binds to a RAGE domain including, but not limited to, the V domain (amino acids 24- 129 of SEQ ID NO : 1 ), the C 1 domain (amino acids 130-234 of SEQ ID NO:1), the C2 domain (amino acids 235-336 of SEQ ID NO:1), the VCl domains (amino acids 24-234 of SEQ ID NO:1), and the C1C2 domain (amino acids 130-336 of SEQ ID NO:1).
  • a polynucleotide of the invention encodes an antibody that binds to the C2 domain (amino acids 235-336 of SEQ ID NO: l) ofRAGE.
  • Nucleic acids of the invention also comprise a nucleotide sequence encoding any of the antibody variable region amino acid sequences shown in SEQ ID NOs:4, 5, 6, 7, 8, and 9. Specific nucleic acids of the invention include but are not limited to SEQ ID NOs: 10- 15. [0096] Nucleic acids of the invention may also comprise a nucleotide sequence encoding an anti-RAGE antibody variable region having an amino acid sequence that is substantially identical to any of the amino acid sequences shown in SEQ ID NOs:4-9, including a nucleotide sequence encoding an amino acid sequence that is at least 85%, 86%,
  • Nucleic acids of the invention may also comprise a nucleotide sequence encoding an anti-RAGE antibody variable region having an sequence that is substantially identical to any of the nucleotide sequences shown in SEQ ID NOs: 10- 15, including a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to any of SEQ ID NOs:10-15.
  • Sequences are compared for maximum correspondence using a sequence comparison algorithm using the full-length variable region encoding sequence of any one of SEQ ID NOs:4-9, a nucleotide sequence encoding a full length variable region having any one of the sequences shown in SEQ ID NOs:4-9, the nucleotide sequence of SEQ ID NOs : 10- 15 as the query sequence, as described herein, or by visual inspection.
  • Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population.
  • An allelic difference may be as small as one base pair.
  • Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • a mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
  • nucleic acids that hybridize specifically to or hybridize substantially to the full length of any nucleotide sequence encoding an antibody variable region amino acid sequence shown in SEQ ID NO:
  • nucleic acid hybridization two nucleic acid sequences being compared may be designated a probe and a target.
  • a probe is a reference nucleic acid molecule
  • a target is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules.
  • a target sequence is synonymous with a test sequence.
  • probes are complementary to or mimic at least about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention.
  • probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any nucleotide sequence encoding an antibody variable region amino acid sequence shown in SEQ ID NOs:4-9, or the nucleotide sequence of SEQ ID NOs: 10-15.
  • fragments may be readily prepared, for example, by chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.
  • stringent hybridization conditions is intended overnight incubation at 42 0 C in a solution comprising: 50% formamide, 5X SSC (750 rnM NaCl, 75 niM trisodium cirate), 50 rnM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65 0 C.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • the amino acid sequence of the heavy and/or light chain variable domains of the antibodies of the invention may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability .
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, J. MoI. Biol. 278 : 457-479 (1998) for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art. [0107] In addition, techniques developed for the production of "chimeric antibodies"
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al, Science 242:1038-1041 (1988)).
  • Antibodies of the invention include derivatives that are modified (i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment).
  • the antibody derivatives include antibodies that have been modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • Antibodies or antigen binding fragments thereof with increased in vivo half- lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG).
  • PEG polymer molecules
  • PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies.
  • Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
  • antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half- life in vivo.
  • the techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413, 622.
  • the present invention encompasses the use of antibodies or antigen binding fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • the present invention encompasses the use of antibodies or antigen binding fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al, 1994, Immunol. Lett. 39:91-99; U.S. Patent No. 5,474,981; Gillies et al, 1992, PNAS 89:1428-1432; and Fell et al, 1991, J. Immunol. 146:2446-2452.
  • the present invention further includes compositions comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments.
  • the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos.
  • EP 307,434 and EP 367,166 International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154:5590-5600; and ViI et al, 1992, Proc. Natl. Acad. Sci. USA 89:11337- 11341.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or antigen binding fragments thereof (e.g. , antibodies or antigen binding fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos.
  • Antibodies or antigen binding fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to an Antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the antibodies or antigen binding fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767) and the "flag" tag.
  • antibodies of the present invention or analogs or derivatives thereof are conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine ( 131 1, 125 1, 123 1, 121 I,), carbon ( 14 C), sulfur ( 35
  • the present invention further encompasses uses of antibodies of the invention or antigen binding fragments thereof conjugated to a therapeutic agent.
  • An antibody or antigen binding fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include ribonuclease, monomethylauristatin E and F, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine
  • alkylating agents e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine
  • CCNU cyclothosphamide
  • busulfan dibromomannitol
  • streptozotocin mitomycin C
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • a more extensive list of therapeutic moieties can be found in PCT publications WO 03/075957.
  • an antibody or antigen binding fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF- ⁇ , TNF- ⁇ , AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No.
  • a toxin such as abrin, ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived
  • a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
  • a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL-I”), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)).
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • G-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • GH growth hormone
  • an antibody can be conjugated to therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials).
  • the macrocyclic chelator is l,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et al., 1998, CHn Cancer Res. 4:2483; Peterson et al, 1999, Bioconjug. Chem. 10:553; and Zimmerman et al, 1999, Nucl. Med. Biol. 26:943.
  • Moieties can be conjugated to antibodies by any method known in the art, including, but not limited to aldehyde/Schiff linkage, sulphydryl linkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage, enzymatically degradable linkage (see generally Garnett, 2002, Adv Drug Deliv Rev 53:171). Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R.
  • linker molecules are commonly known in the art and described in Denardo et al., 1998, CHn Cancer Res 4:2483; Peterson et al, 1999, Bioconjug Chem 10:553; Zimmerman et al, 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the therapeutic moiety or drug conjugated to an Fc variant of the invention should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject. A clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to an Fc variant of the invention: the nature of the disease, the severity of the disease, and the condition of the subject.
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • an antibody of the invention or antigen binding fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, 3T3, PerC6 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promote
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
  • U.S. patents 5827739, 5879936, 5981216, and 5658759 are examples of cells.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome ⁇ e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts ⁇ e.g., see Logan & Shenk, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications ⁇ e.g., glycosylation) and processing ⁇ e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, NSO, Per.C ⁇ and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst.
  • breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D
  • normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst.
  • stable expression is preferred.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, Cell 11 :223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Proc Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., MoI. Cell. Biol. 3:257 (1983)).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility
  • the antibodies of the present invention or antigen binding fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
  • the antibodies or antigen binding fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag” tag.
  • the antibodies of the invention may be assayed for specific (i.e., immunospecific) binding by any method known in the art.
  • the immunoassays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, cell surface binding assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4 0 C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4 0 C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the anti
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York at 11.2.1.
  • Cell surface binding assays comprise identifying or generating a cell which displays the antigen on its cell surface, adding the antibody of interest to the cell and incubating for a period of time, and detecting the presence of the antibody on the cells surface. Such detection generally employs methods such as fluorescence activated cell sorting (FACS) analysis.
  • FACS fluorescence activated cell sorting
  • the antibodies to be tested may either directly coupled to a detectable compound such as a fluorescent dye, or alternative may be detected using a secondary antibody coupled to a detectable compound.
  • the binding affinity and other binding properties (e.g., off-rate of an antibody- antigen interaction) of an antibody to an antigen may be determined by a variety of in vitro assay methods well known in the art including for example, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE ® analysis), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • equilibrium methods e.g., enzyme-linked immunoabsorbent assay (ELISA; or radioimmunoassay (RIA)
  • kinetics e.g., BIACORE ® analysis
  • indirect binding assays e.g., competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration
  • binding affinities and kinetics can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.
  • the antibodies of the invention may be assayed for biological activity by any method known in the art, and as disclosed herein, see the Examples Section 6.2.
  • the protocols and formulations of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays which can be used to determine whether administration of a specific therapeutic protocol formulation or combination therapy of the invention is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise contacted with a formulation of the invention, and the effect of such a formulation upon the tissue sample is observed.
  • the tissue sample can be obtained by biopsy from the patient.
  • in vitro assays can be carried out with representative cells of cell types involved in an autoimmune disorder, an inflammatory disorder, a disorder mediated by RAGE and/or HMGBl, a disorder characterized by the presence of DNA immune complexes, a disorder TNF-alpha mediated inflammation, to determine if a formulation of the invention has a desired effect upon such cell types.
  • a formulation of the invention has a desired effect upon such cell types.
  • a lower level of expression of one or more type-I IFN -inducible gene and/or a lower level of proinflammatory cytokines produced by the contacted cells indicates that the composition of the invention may be effective to treat the disorder or condition in the patient.
  • a formulation of the invention may be screened using cells which can be stimulated to stimulate the expression of one or more type-I IFN -inducible gene such as for example peripheral blood mononuclear cells (PBMCs), THP-I cells or Macrophages (M0s).
  • PBMCs peripheral blood mononuclear cells
  • M0s Macrophages
  • Many assays standard in the art can be used to assess IFN-inducible gene expression and/or cytokine production including ELISA assays, realtime PCR. Such methods are well known in the art and exemplified herein, see the Examples Section 6.2.
  • Prophylactic or therapeutic agents can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc.
  • compositions of the invention comprise one or more antibody of the invention in a pharmaceutically acceptable excipient.
  • pharmaceutically-acceptable carrier means a chemical composition with which an antibody of the invention may be combined and which, following the combination, can be used to administer the antibody of the invention to a subject.
  • the present invention further includes methods for treating a disorder or condition characterized by TNF-alpha mediated inflammation and or conditions characterized by the presence of DNA immune complexes, including both acute and chronic inflammatory conditions, comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition of the invention.
  • Chronic inflammatory conditions are characterized by an inflammatory response of prolonged duration - weeks, months, or even indefinitely which results in tissue damage that is often permanent.
  • Acute inflammatory conditions are usually characterized by a sudden onset of symptoms including, increased vascular permeability, oedema, systemic fever often resulting in tissue necrosis and may result in death.
  • compositions of the invention comprise antibodies that bind to RAGE (e.g., an epitope within the polypeptide encoded by GenBankTM accession number BC020669).
  • compositions comprise antibodies of the invention that bind to the C2 domain of RAGE (e.g., an epitope within amino acids 234-342 or amino acids 235-336 of SEQ ID NO: 1).
  • compositions of the invention can comprise the antibodies of the invention alone or in combination with other active therapeutic molecules and/or adjuvants such as steroids, other anti-inflammatory molecules, or other antibody therapeutics.
  • the present invention further provides methods of preventing, managing, treating or ameliorating a disorder, or one or more symptoms thereof, including, but not limited to, a disorder characterized by TNF-alpha mediated inflammation and/or by the presence of DNA immune complexes and/or an HMGBl -mediated disorder or condition, said methods comprising administering to a subject in need thereof an effective amount of an antibody composition of the invention.
  • the methods also encompass the administration, to a subject in need thereof, an effective amount of an antibody composition of the invention in combination with an addition therapeutic agent.
  • Any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of a disorder, characterized by TNF-alpha mediated inflammation and/or by the presence of DNA immune complexes and/or an HMGBl -mediated disorder or condition can be used in combination with an antibody composition of the invention.
  • agents include, but are not limited to, immunomodulatory agents, an anti- angiogenic agents, anti-inflammatory agents and TNF ⁇ antagonists.
  • the present invention provides a method for treating an HMGBl -mediated disorder or condition, comprising administering an effective amount of an antibody composition of the invention.
  • HMGBl -mediated disorders or conditions include, but are not limited to, systemic lupus erythematosus, inflammatory lupus nephritis psoriasis, Sjogren's disease, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, COPD, scleroderma, ulcerative colitis, inflammatory bowel disease, type I diabetes, allograft rejection and graft-versus-host disease.
  • the present invention provides a method for treating a RAGE -mediated disorder or condition, comprising administering an effective amount of an antibody composition of the invention.
  • RAGE -mediated conditions or disorders may be characterized generally as including any disorder in which an affected cell exhibits elevated expression of RAGE and/or one or more RAGE ligands.
  • RAGE -mediated conditions or disorders may also be characterized as any disorder that is treatable (i.e., one or more symptoms may be eliminated or ameliorated) by a decrease in RAGE function.
  • RAGE function can be decreased by administering an agent that disrupts the interaction between RAGE and one or more of its ligands.
  • RAGE ligands are produced in tissue affected with many inflammatory disorders, including arthritis (e.g., rheumatoid arthritis). In diabetic tissues, the production of RAGE is thought to be caused by the overproduction of advanced glycation end products (AGEs). This overproduction results in oxidative stress and endothelial cell dysfunction that leads to vascular disease in diabetics (WO 2004/016229; the entire teachings of which are incorporated herein by reference).
  • AGEs advanced glycation end products
  • amyloidoses binds to beta-sheet fibrillar material, such as that found in amyloid-beta peptide, Abeta, amylin, serum amyloid A and prion-derived peptides. Increased expression of RAGE is also observed in tissues having amyloid structures. Accordingly, RAGE is involved in amyloid disorders. The RAGE-amyloid interaction is thought to result in oxidative stress leading to neuronal degeneration (WO 2004/016229).
  • the present invention provides a method for treating a subject having a condition or disorder characterized by amyloid deposit of amyloid- ⁇ -peptide (A ⁇ ), such as Alzheimer's disease, which comprises administering to a subject a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular A ⁇ .
  • a ⁇ amyloid- ⁇ -peptide
  • the invention also provides a method of inhibiting or reducing accumulation of amyloid deposit of A ⁇ in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner. Also included within the invention is a method of inhibiting or reducing neurodegeneration in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner. The invention further includes a method of inhibiting or reducing cognitive decline, or improving cognition, in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner.
  • the invention also provides a method for treating a subject having an amyloidogenic disease or disorder characterized by amyloid deposit which comprises administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular A ⁇ .
  • the present invention provides a method for treating a subject having a condition or disorder characterized by amyloid deposit of A ⁇ , such as Alzheimer's disease, which comprises administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular A ⁇ , to a subject under conditions that generate a beneficial therapeutic response in the subject (e.g., reduction of plaque burden, inhibition of plaque formation, reduction of neuritic dystrophy, and improvement of cognitive function, e.g., rapidly improving cognition, and/or reversing, treating or preventing cognitive decline).
  • a beneficial therapeutic response in the subject e.g., reduction of plaque burden, inhibition of plaque formation, reduction of neuritic dystrophy, and improvement of cognitive function, e.g., rapidly improving cognition, and/or reversing, treating or preventing cognitive decline.
  • a ⁇ amyloid deposits of A ⁇ in the brain
  • diseases, disorders or conditions associated with amyloid deposits of A ⁇ in the brain include Alzheimer's disease, Down's syndrome and cognitive impairment. The latter can occur with or without other characteristics of an amyloidogenic disease.
  • the ligands of RAGE include proteins having ⁇ -sheet fibrillar structures that are characteristic of amyloid deposits and pro-inflammatory mediators, including beta-amyloid protein (A ⁇ ), serum amyloid (SAA) (fibrillar form),
  • SlOO/calgranulins e.g., S100A12, SlOOB, S100A8-A9
  • HMGBl high mobility group box-1 chromosomal protein 1
  • SAA serum amyloid A
  • RAGE is associated with the accumulation of amyloid in kidneys and the tissue destruction leading to kidney failure of individuals with familial amyloidotic polyneuropathy (FAP) (Matsunaga et al., 2005, Scand. J. Clin. Lab. Invest.).
  • the RAGE ligand amphoterin (HMGBl) also contains an amyloidogenic peptide, one that is highly homologous to the Alzheimer's A ⁇ peptide and forms amyloid-like peptides when released from the native protein (Kallijarvi et al., 2001, Biochem., 40:10032-7).
  • a ⁇ The interaction of A ⁇ with RAGE-bearing cells in the walls of blood vessels results in transport of A ⁇ across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-I), the latter mediating A ⁇ -induced vasoconstriction.
  • BBB blood-brain barrier
  • ET-I proinflammatory cytokines and endothelin-1
  • the present invention also provides methods for reducing A ⁇ -induced vasoconstriction.
  • RAGE -ligand interaction has been shown to suppress the accumulation of A ⁇ in brain parenchyma in a transgenic mouse model for Alzheimer's-like disease (Deane et al., 2003, Nature Medicine 9:907-913).
  • the active, pathogenic role of RAGE in a wide range of amyloidogenic diseases, disorders, and conditions makes it possible to provide therapeutic, beneficial treatment to patients with these amyloidogenic disorders by the method of the present invention, which provides antibodies that bind specifically to RAGE and inhibit the binding of a RAGE binding partner.
  • Patients amenable to treatment by the invention include individuals at risk of an A ⁇ -related disease or disorder or amyloidogenic disease or disorder but not showing symptoms, as well as patients presently showing symptoms.
  • Alzheimer's disease virtually anyone is at risk of suffering from Alzheimer's disease if he or she lives long enough. Therefore, the present methods can be administered prophylactically to the general population without the need for any assessment of the risk of the subject patient.
  • the present methods are especially useful for individuals who are at risk for Alzheimer's disease, e.g., those who exhibit risk factors of Alzheimer's disease. Thus, the present methods are useful for preclinical Alzheimer's disease.
  • the main risk factor for Alzheimer's disease is increased age. As the population ages, the frequency of Alzheimer's disease continues to increase. Current estimates indicate that up to 10% of the population over the age of 65 and up to 50% of the population over the age of 85 have Alzheimer's disease.
  • the methods of the invention can be used on both asymptomatic patients and those currently showing symptoms of disease.
  • the antibodies used in such methods can be human, humanized, chimeric or nonhuman antibodies, or antigen binding fragments thereof (e.g., RAGE binding fragments), as described herein.
  • the present invention provides a method for inhibiting or reducing cognitive decline, and/or improving cognition, in a patient having or at risk for suffering from an A ⁇ - related disease, disorder or condition or amyloidogenic disease or disorder (e.g., Alzheimer's disease), comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular A ⁇ .
  • an A ⁇ - related disease, disorder or condition or amyloidogenic disease or disorder e.g., Alzheimer's disease
  • Cognition in humans may be assessed using a wide variety of tests including, but not limited to, the following tests: ADAS-Cog (Alzheimer Disease Assessment Scale- Cognitive) (Rosen et al. (1984) Am J Psychiatry. 141(11):1356-64; IhI et al. (2000) Neuropsychobiol. 41(2): 102-7; and Weyer et al. (1997) Int Psychogeriatr. 9(2): 123-38);
  • Screen Test Blessed et al. (1968) Br J Psychiatry 114(512):797-811
  • MMSE Mini Mental State Exam
  • MMSE Felstein et al. (1975) J Psychiatr Res. 12:196-198; Cockrell & Folstein (1988) Psychopharm Bull. 24(4):689-692; and Crum et al. (1993) J. Am. Med. Association 18:2386-2391); Seven- Minute Screen (Solomon & Pendlebury (1998) Fam Med. 30(4):265-71, Solomon et al.
  • AD Alzheimer's disease and Related Disorders Association
  • the anti-RAGE antibodies of the present invention may be used in combination with one or more additional agents, which may be administered to a subject concurrently or sequentially in either order.
  • the disclosed combination therapies may elicit a synergistic therapeutic effect, Le, an effect greater than the effect of either agent alone.
  • a synergistic therapeutic effect may be an effect of at least about two-fold greater than the therapeutic effect elicited by a single agent, or at least about at least about five-fold greater, or at least about ten-fold greater, or at least about twenty-fold greater, or at least about fifty-fold greater, or at least about one hundred-fold greater.
  • the invention includes administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner in combination with another antibody that binds specifically to A ⁇ .
  • the antibody that binds to A ⁇ can be an antibody that specifically binds to A ⁇ peptide without binding to full-length amyloid precursor protein (APP).
  • the antibody of the invention may be administered in combination with antibodies that bind to and/or capture soluble A ⁇ , or that bind to an amyloid deposit in the patient and induce a clearing response against the amyloid deposit. Such a clearing response can be effected by Fc receptor mediated phagocytosis.
  • Such a clearing response can be engineered into an antibody, for example, by including an Fc receptor-binding domain (e.g., an IgG2a constant region).
  • the antibody of the invention can also be administered to a patient who has received or is receiving an A ⁇ vaccine.
  • antibodies of the invention can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier.
  • Antibodies of the invention can also be administered in combination with other agents that enhance access of the therapeutic agent to a target cell or tissue, for example, liposomes and the like. Coadministering such agents can decrease the dosage of a therapeutic agent (e.g., therapeutic antibody or antibody chain) needed to achieve a desired effect.
  • a therapeutic agent e.g., therapeutic antibody or antibody chain
  • a variety of RAGE ligands are produced in inflamed tissues. This observation is true both for acute inflammation, such as that seen in response to a lipopolysaccharide challenge (as in sepsis), and for chronic inflammation. Cardiovascular diseases, particularly those arising from atherosclerotic plaques, are thought to have a substantial inflammatory component and therefore can be treated using the compositions of the invention. Such cardiovascular diseases include, e.g., occlusive, thrombotic and embolic diseases, such as angina, fragile plaque disorder and embolic stroke, respectively, as well as the other cardiovascular diseases described herein
  • the present invention provides a method for treating TNF-alpha mediated inflammation and/or a disorder characterized by TNF-alpha mediated inflammation.
  • the present invention provides a method for treating a disorder characterized by the presence of DNA immune complexes, including, but not limited to, SLE.
  • the prophylactic or therapeutic agents used in combination with a composition of the invention can also be cyclically administered.
  • Cycling therapy involves the administration of a first prophylactic or therapeutic agent for a period of time, followed by the administration of a second prophylactic or therapeutic agent for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the agents, to avoid or reduce the side effects of one of the agents, and/or to improve the efficacy of the treatment.
  • disorders or conditions which can be usefully treated using the antibody compositions, i.e., pharmaceutical compositions of the present invention include those disorders or conditions enumerated in the background section of this specification and below.
  • the disorder or condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis,
  • the disorder or condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, scleroderma, COPD, disseminated bacteremia, burns, preclinical Alzheimer's disease, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, SCI, paralysis, SLE, allograft rejection or graft-versus-host disease.
  • a “therapeutically effective amount,” an “amount sufficient” and like terms refers to that amount of the therapeutic agent, e.g., a RAGE antibody composition of the invention, sufficient to treat or manage a disease or disorder for example, a disorder or condition characterized by TNF-alpha mediated inflammation and/or the presence (or elevated levels) of DNA immune complexes.
  • a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of the disease, e.g., delay or minimize the severity of a disease.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of an inflammatory disorder.
  • a therapeutically effective amount with respect to a pharmaceutical composition of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management a disease, e.g., an inflammatory disease.
  • a therapeutically effective dose preferably prevents further deterioration of physical symptoms associated with SLE, such as, for example, pain, fatigue or weakness.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • the invention is directed to methods of administering and using compositions and antibodies or the invention to treat and/or prevent a disorder or condition selected from the group consisting of appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, hepatitis, Crohn's disease, COPD, scleroderma, asthma, allergy, anaphylactic shock, rheumatoid arthritis, SLE, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, disseminated bacteremia, burns, rheumatoid arthritis, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection and graft-versus-host disease, type I diabetes.
  • a disorder or condition selected
  • Certain embodiments of the invention are directed to methods of administering and using compositions and antibodies of the invention to treat and/or prevent sepsis, lupus and arthritis (e.g., RA, psoriatic arthritis, juvenile rheumatoid arthritis).
  • a specific embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and prevent lupus.
  • the antibody compositions e.g., pharmaceutical compositions of the invention, reduce the TNF-alpha mediated inflammation in a mammal by at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90% as compared to a control composition.
  • the antibody compositions i.e., pharmaceutical compositions of the invention reduce a disease index score (e.g., Systemic Lupus Activity Measure (SLAM), Systemic Lupus Erythematosus Disease Activity Index
  • SLAM Systemic Lupus Activity Measure
  • SAM Systemic Lupus Erythematosus Disease Activity Index
  • SLEDAI SLEDAI in a mammal suffering from lupus by at least 5%, or at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90% as compared to a control composition.
  • any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of an inflammatory disorder or one or more symptoms thereof can be used in combination with an antibody composition of the invention.
  • anti-CD19 antibodies ⁇ see, e.g., U.S. and international Patent Publications US20020041847, US20030133930 and WO 05/012493
  • anti-CD5 antibodies e.g., an anti-CD5 ricin-linked immunoconjugate
  • anti-CD7 antibodies e.g., CHH-380 (Novartis)
  • anti-CD8 antibodies anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)
  • anti-CD52 antibodies e.g., CAMPATH IH (Ilex)
  • anti-CD2 antibodies e.g., MEDI-507 (Medlmmune, Inc., International Publication Nos.
  • anti-CD 1 Ia antibodies e.g., Xanelim (Genentech)
  • anti-B7 antibodies e.g., IDEC-114)
  • anti-cytokine receptor antibodies e.g., anti- IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)
  • anti-IL-4 receptor antibodies e.g., anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies
  • anti-cytokine antibodies e.g., anti-IFN antibodies, anti- TNF- ⁇ antibodies, anti-IL- ⁇ antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX- IL-8 (Abgenix)
  • anti-CD22 antibodies e.g., non- ligand blocking antibodies such as Epratuzumab (Immunomedics) and ligand blocking antibodies (e.g., U.S.
  • IL interleukin
  • IFN interferon
  • anti-cytokine antibodies e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF- ⁇ antibodies, and anti-IFN-. gamma. antibodies).
  • Non-limiting examples of anti-angiogenic agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include Vitaxin® (Medlmmune) or other anti-alpha v beta3 antibodies (e.g., CNTO95 (Centocor)), endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of platelet factor-4, the anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the
  • Non-limiting examples of TNF- ⁇ antagonists which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab) 2 fragments, and antigen-binding fragments thereof) such as antibodies that immunospecifically bind to TNF- ⁇ , nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules that blocks, reduces, inhibits or neutralizes the function, activity and/or expression of TNF- ⁇ .
  • proteins e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab) 2 fragments, and antigen-binding fragments thereof
  • nucleic acid molecules e.g.
  • a TNF- ⁇ antagonist reduces the function, activity and/or expression of TNF- ⁇ by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a control such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • antibodies that immunospecifically bind to TNF- ⁇ include, but are not limited to, infliximab (REMICADETM; Centocor), D2E7 (Abbott)
  • the present invention also encompasses the use of antibodies that immunospecifically bind to TNF- ⁇ disclosed in the following U.S. patents in the compositions and methods of the invention: U.S. Pat. Nos.
  • soluble TNF- ⁇ receptors include, but are not limited to, sTNF-Rl (Amgen), etanercept (ENBRELTM; Immunex) and its rat homo log RENBRELTM, soluble inhibitors of TNF- ⁇ derived from
  • TNFrI TNFrII
  • TNFrII Kohno et al, 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335
  • TNF- ⁇ Inh Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192.
  • TNF- ⁇ antagonists encompassed by the invention include, but are not limited to, IL-10, which is known to block TNF- ⁇ production via interferon .gamma.- activated macrophages (Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89:8676-8680),
  • TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-I (Serono/Yeda), the vaccine CytoTAb (Protherics), antisense molecule 104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC- 333 (Dupont), VX-745 (Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco), NPI- 13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax), auranofm (Ridaura) (SmithKline Beecham Pharmaceuticals), quinacrine (mepacrine dichlor
  • Non-limiting examples of anti-inflammatory agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-agonists, anticholingeric agents, and methyl xanthines.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • beta-agonists beta-agonists
  • anticholingeric agents methyl xanthines
  • NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREXTM), diclofenac (VOLTARENTM), etodolac (LODINETM), fenoprofen (NALFONTM), indomethacin (INDOCINTM), ketoralac (TORADOLTM), oxaprozin (DAYPROTM), nabumentone (RELAFENTM), sulindac (CLINORILTM), tolmentin (TOLECTINTM.), rofecoxib (VIOXXTM), naproxen (ALEVETM, NAPROSYNTM), ketoprofen (ACTRON) and nabumetone (RELAFENTM).
  • NSAIDs function by inhibiting a cyclooxgenase enzyme (e.g., COX-I and/or COX-2).
  • a cyclooxgenase enzyme e.g., COX-I and/or COX-2
  • steroidal anti- inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRONTM), cortisone, hydrocortisone, prednisone (DELTASONETM), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes, and leukotrienes.
  • patients with osteoarthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful for osteoarthritis prevention, treatment, management or amelioration including but not limited to: analgesics (non- limiting examples are acetaminophen, in a dose up to 4000 mg/d; phenacetin; and tramadol, in a daily dose in the range of 200 to 300 mg); NSAIDs (non- limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.
  • analgesics non- limiting
  • NSAIDs Low dose NSAIDs are preferred, e.g., ibuprofen at 1200 mg/d, naproxen at 500 mg/d.
  • a gastroprotective agent e.g., misoprostol, famotidine or omeprazole, is preferred to use concurrently with a NSAID); nonacetylated salicylates including but not limited to salsalate; cyclooxygenase (Cox)-2-specific inhibitors (CSIs), including but not limited to, celecoxib and rofecoxib; intra- or periarticular injection of a depot glucocorticoid preparation; intra-articular injection of hyaluronic acid; capsaicin cream; copious irrigation of the osteroarthritis knee to flush out fibrin, cartilage shards and other debris; and joint replacement surgery.
  • Cox-2-specific inhibitors including but not limited to, celecoxib and rofecoxib
  • the antibody compositions of the invention can also be used in combination with other nonpharmacologic measures in prevention, treatment, management and amelioration of osteoarthritis including but not limited to: reduction of joint loading (non- limiting examples are correction of poor posture, support for excessive lumbar lordosis, avoid excessive loading of the involved joint, avoid prolonged standing, kneeling and squatting); application of heat to the affected joint; aerobic exercise and other physical therapies.
  • patients with rheumatoid arthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to: NSAIDs (non-limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.); analgesics (non-limiting examples are acetaminophen, phenacetin and tramadol); CSIs including but not limited to, celecoxib and rofecoxi
  • the antibody compositions of the invention may also be used in combination with other measures in prevention, treatment, management and amelioration of the rheumatoid arthritis including but not limited to: rest, splinting to reduce unwanted motion of inflamed joint, exercise, used of a variety of orthotic and assistive devices, and other physical therapies.
  • the antibody compositions of the invention may also be used in combination with some nontraditional approaches in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to, diets (e.g., substituting omega-3 fatty acids such as eicosapentaenoic acid found in certain fish oils for dietary omega-6 essential fatty acids found in meat), vaccines, hormones and topical preparations.
  • diets e.g., substituting omega-3 fatty acids such as eicosapentaenoic acid found in certain fish oils for dietary omega-6 essential fatty acids found in meat
  • vaccines e.g., eicosapentaenoic acid found in
  • COPD are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with other agents or therapies useful in prevention, treatment, management and amelioration of COPD including, but not limited to: bronchodilators including but not limited to, short- and long- acting ⁇ 2 -adrenergic agonists (examples of short-acting ⁇ 2 agonist include but not limited to, albuterol, pirbuterol, terbutaline, and metaproterenol; examples of long-acting ⁇ 2 agonist include but not limited to, oral sustained-release albuterol and inhaled salmeterol), anticholinergics (examples include but not limited to ipratropium bromide), and theophylline and its derivatives (therapeutic range for theophylline is preferably 10-20 ⁇ g/mL); glucocorticoids; exogenous ⁇ iAT (e.g., (Xi AT derived from pooled human plasma administered intravenously in
  • patients with pulmonary fibrosis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for pulmonary fibrosis therapy including but not limited to: oxygen; corticosteroids (a non- limiting example is to administer daily prednisone beginning at 1-1.5 mg/kg/d (up to 100 mg/d) for six weeks and tapering slowly over 3-6 months to a minimum maintenance dose of 0.25 mg/kg/d); cytotoxic drugs (non-limiting examples are cyclophosphamide at 100-120 mg orally once daily, and azathioprine at 3 mg/kg up to 200 mg orally once daily); bronchodilators (non-limiting examples are short- and long- acting ⁇ 2 -adrenergic agonists, anticholinergics, and theophylline and its derivatives); and antihistamines (non- limiting examples are diphenhydramine and doxyl
  • patients with SCI are administered prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for SCI therapy including but not limited to: glucocorticoid steroids (a non-limiting example is to administer methylprednisolone 30 mg/kg bolus over 15 minutes and an infusion of methylprednisolone at 5.4 mg/kg/h for 23 hours beginning 45 minutes after the bolus), neuroprotectors (e.g., minocyclin), regeneration therapies (e.g., stem cell treatments, hydrogels), weak electrical fields (e.g., extraspinal oscillating field stimulator implantable medical device).
  • glucocorticoid steroids e.g., to administer methylprednisolone 30 mg/kg bolus over 15 minutes and an infusion of methylprednisolone at 5.4 mg/kg/h for 23 hours beginning 45 minutes after the bolus
  • neuroprotectors e.g., mino
  • patients with asthma are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for asthma therapy including but not limited to: adrenergic stimulants (examples include but not limited to, catecholamines, e.g., epinephrine, isoproterenol, and isoetharine; resorcinols, e.g., metaproterenol, terbutaline, and fenoterol; and saligenins, e.g., salbutamol.
  • adrenergic stimulants include but not limited to, catecholamines, e.g., epinephrine, isoproterenol, and isoetharine
  • resorcinols e.g., metaproterenol, terbutaline, and fenoterol
  • saligenins e.g., salbutamol.
  • methylxanthines including but not limited to theophylline and its various salts
  • anticholinergics including but not limited to, atropine sulfate, atropine methylnitrate, and ipratropium bromide
  • glucocorticoids examples including but not limited to systemic or oral steroids, and inhaled glucocorticoids
  • mast cell stabilizing agents include but not limited to, cromolyn sodium and nedocromil sodium
  • leukotriene modifiers include but not limited to, Zileuton, zafirlukast and montelukast
  • immunosuppressant agents include but not limited to, methotrexate and gold salts
  • mucolytic agents examples include but not limited to acetylcysteine
  • the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
  • the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side effects).
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • the pharmaceutical compositions of the invention are pyrogen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • the compositions described herein should be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes or by other means well known in the art. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington's Pharmaceutical Sciences (18 th ed., Mack Publishing Company, Easton, PA, 1990).
  • compositions comprising antibodies ordinarily will be stored in lyophilized form or in solution. It is contemplated that sterile compositions comprising antibodies of the invention are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • the composition of the present invention may be administered orally, parenterally, i.e., including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques, by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • said composition in accordance with the method of the present invention, can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • the present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
  • Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
  • Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et ah, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid) [0205]
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sd. Rev. Macromol. Chem.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • the present invention also provides pharmaceutical compositions.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
  • human antibodies have a longer half- life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, Hpidation.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the excipient included with the polypeptide in these compositions is chosen based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the disorder or condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as endotoxic shock, and oral administration may be preferred to treat a gastrointestinal disorder such as a gastric ulcer.
  • the route of administration and the dosage of the composition to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the disorder/condition or disorders/conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the antibody composition can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabuccaly and transdermally to the patient.
  • compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier.
  • the compositions may be enclosed in gelatin capsules or compressed into tablets.
  • the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
  • Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents.
  • binders include microcrystalline cellulose, gum tragacanth or gelatin.
  • excipients include starch or lactose.
  • disintegrating agents include alginic acid, corn starch and the like.
  • lubricants include magnesium stearate or potassium stearate.
  • An example of a 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.
  • compositions of the present invention can easily be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection.
  • Parenteral administration can be accomplished by incorporating the antibody compositions of the present invention into a solution or suspension.
  • 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.
  • Rectal administration includes administering the pharmaceutical compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art.
  • suppository formulations can be prepared by heating glycerin to about 120C, dissolving the antibody composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
  • Transdermal administration includes percutaneous absorption of the composition through the skin.
  • Transdermal formulations include patches, ointments, creams, gels, salves and the like.
  • the antibody compositions described herein can also include an antagonist of an early sepsis mediator.
  • an early sepsis mediator is a proinflammatory cytokine that is released from cells soon (i.e., within 30-60 min.) after induction of an inflammatory cytokine cascade (e.g., exposure to LPS).
  • Nonlimiting examples of these cytokines are TNF, IL-l ⁇ , IL-l ⁇ , IL-6, PAF, and MIF.
  • receptors for these cytokines for example, tumor necrosis factor receptor type 1
  • enzymes required for production of these cytokines for example, interleukin-l ⁇ converting enzyme.
  • Antagonists of any early sepsis mediator can be useful for these embodiments by further inhibiting an inflammatory cytokine cascade.
  • Nonlimiting examples of antagonists of early sepsis mediators are antisense compounds that bind to the mRNA of the early sepsis mediator, preventing its expression (see, e.g., Ojwang et al., 1997, Biochemistry 36:6033-6045; Pampfer et al., 1995, Biol. Reprod. 52:1316-1326; U.S. Patent No. 6,228,642; Yahata et al, 1996, Antisense Nucleic Acid Drug Dev. 6:55-61; and Taylor et al., 1998, Antisense Nucleic Acid Drug Dev. 8:199-
  • Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention.
  • the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
  • the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art ⁇ e.g., see Jalkanen, et al., 1985, J. Cell. Biol. 101 :976-985; Jalkanen, et al, 1987, J. Cell. Biol.
  • antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003).
  • diagnosis comprises: (a) administering (for example, parenterally, subcutaneous Iy, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; (b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); (c) determining background level; and (d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
  • Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined
  • antibodies of the invention may be used to treat, diagnose, or prognose an individual having sepsis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., RA, psoriatic arthritis, juvenile rheumatoid arthritis), anaphylactic shock, organ ischemia, reperfusion injury, and allograft rejection, systemic lupus erythematosus, Type I diabetes and related pathological states, including diabetic vasculopathy, nephropathy, retinopathy, neuropathy, Alzheimer's disease and immune/inflammatory reactions of blood vessel walls and other RAGE -mediated conditions or disorders.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S. W. Burchiel et ah, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours.
  • the time interval following administration is 5 to 20 days or 5 to 10 days.
  • monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et ah, U.S. Pat. No. 5,441,050).
  • the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
  • the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • said antibody inhibits the binding of an HMGB 1 -CpG complex to RAGE.
  • said antibody comprises a heavy chain variable region selected from the group consisting of the antibody S2, SEQ ID NO:5, antibody L30, SEQ ID NO:7, and the antibody L37, SEQ ID NO:9.
  • [0256] 15 The antibody of any of the preceding embodiments, wherein said antibody inhibits mammalian cell RAGE-mediated expression of one or more Type I Interfereon (IFN) genes or one or more type-I IFN-inducible genes selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35.
  • IFN Type I Interfereon
  • TLR is selected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRIl.
  • TLRl is selected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRIl.
  • the antibody is selected from the group consisting of: a human antibody, a humanized antibody; a chimeric antibody; a single-chain Fv (scFv); an Fab fragment; an F(ab') fragment; an intrabody; and a synthetic antibody.
  • An antibody comprising at least one CDR derived from an antibody of embodiment 20, wherein said antibody binds the human RAGE polypeptide of SEQ ID NO: 1 and inhibits the binding of an HMGBl-CpG complex to RAGE.
  • [0266] 25 A composition comprising an antibody of any one of embodiments 1 to 24, and a pharmaceutically acceptable excipient.
  • [0267] 26 A method of reducing TNF-alp ha mediated inflammation in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • a method of treating rheumatoid arthritis in a subject comprising administering an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • a method for treating a condition characterized by the increased presence of DNA immune complexes in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • a method for treating systemic lupus erythematosus in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • [0273] 32 A method for reducing RAGE-mediated expression of one or more type-I IFN-inducible gene selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35 in a subject, comprising administering to the subject an effective amount of a composition comprising the antibody of any one of embodiments 1 to 24.
  • 33 A method for treating an HMGB 1 -mediated condition in a subj ect, comprising administering an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • HMGB 1 -mediated condition is selected from the group consisting of, sepsis, systemic lupus erythematosus, inflammatory lupus nephritis, scleroderma, COPD, psoriasis, Sjogren's disease, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, type I diabetes, allograft rejection and graft-versus-host disease.
  • a method for treating a disease or disorder characterized by amyloid deposit of A-beta in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • A-beta in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
  • [0291] 50 A method of inhibiting or reducing cognitive decline, or improving cognition, in a subject, comprising administering to the subject an effective amount of the antibody of any one of embodiments 1 to 24.
  • Antibody Generation The extracellular domain of human RAGE (huRAGE) fused to Fc region of human IgGl was expressed in NSO cells and used to immunize BALB/c mice. Three groups of mice were immunized as follows: Group I - footpad immunization using TiterMax adjuvant; Group II - i.p. immunization using CFA/IFA adjuvant; Group III - i.p. immunization using TiterMax adjuvant. Hybridomas were generated using standard methodologies.
  • Clones were initially screened for IgG reactivity specific for the huRAGE-Fc fusion protein used for immunization in an ELISA format followed by FACS studies using cells stably (NSO) or transiently (293F) transfected with full-length huRAGE. Positive clones expressing anti-RAGE mAbs were then further purified by limited dilution cloning. The hybridomas are grown in DMEM/2% ultra low IgG serum and the mAbs are purified by protein G chromatography.
  • Bindins Studies and Epitope Mapping The cross-reactivity of anti-huRAGE mAbs against murine RAGE (mRAGE) was examined by ELISA. Epitope mapping of anti- RAGE mAbs was performed using 293F cells expressing the different human RAGE domain deletion mutants shown in Figure 2: V domain (amino acids 1-121), Cl domain (amino acids 122-233), C2 domain (amino acids 234-342), VCl domains (amino acids 1-233), and C1C2 domains (amino acids 122-342) of GenBank accession number BC020669.
  • the signal sequence (amino acids 1-26) was added to the N- terminal of Cl, C2, and C1C2 constructs, and the transmembrane-cytoplasmic tail sequence (amino acids 343-404) was included or added to the C-terminal of all constructs.
  • 293F cells expressing huRAGE and its mutants were incubated with anti-huRAGE mAbs, followed by PE-conjugated goat anti-mouse IgG Fc-specif ⁇ c Fab fragments and analyzed by FACS. Representative data are shown in Figure 3 and summarized in Table 2.
  • HMGBl/CpG complex at 1 : 1 molar ratio was then added to the wells at a final concentration of 30 nM (or 0.8 mg/ml) and incubated at 37 0 C for 1 hr. The wells were then washed, and incubated with 3 nM (or 0.5mg/ml) of biotinylated anti-HMGBl antibody (S16-biotin), followed by detection with streptavidin-HRP. Representative data are shown in Figure 4 and summarized in Table 2 and Table 5. [0300] Surface Plasmon Resonance Measurements: All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden).
  • the ligand, human RAGE/Fc was prepared at 5 ug/mL in 1OmM NaOAc, pH4 buffer, then injected onto an EDC/NHS-activated CM5 sensor chip (BIAcore, Inc. Uppsala, Sweden) using a standard immobilization protocol. Following this, unreacted active ester moieties were quenched by injecting IM Et-NH2 (ethanolamine) over the sensor chip surface. These coupling reagents were also purchased from the manufacturer (BIAcore, Inc.). A total of 573 resonance units (RUs) of huRAGE/Fc remained bound to the sensor chip surface after the coupling procedure. Separately, a blank surface was also prepared on the sensor chip using the identical protocol, minus the protein.
  • IgGs were prepared as two or three fold dilution series, as follows: L30 (0.0391 nM - 40 nM) in HBS-EP buffer (BIAcore, Inc., consisting of the following: 1OmM HEPES buffer, pH7.4, 15OmM NaCl, 3mM EDTA, and 0.005% P20). A duplicate injection of each concentration of the IgGs was then injected over both the huRAGE/Fc and reference cell surfaces, which were connected in series. Between injections, the bound IgG was removed from the huRAGE-Fc surface with two, one-minute injections of 1O mM GIy buffer, pHl.5.
  • a huRAGE-Fc fusion construct was used to generate anti-huRAGE antibodies.
  • 21 anti-huRAGE mAbs were identified. Several were found to have low expression levels and were not further characterized.
  • the anti-huRAGE reactivity of 16 antibodies was characterized by ELISA and FACS analysis. At least one anti-huRAGE antibody cross-reacts with mRAGE by ELISA.
  • the reactive domains of the anti-huRAGE mAbs have been determined by FACS analysis of anti-huRAGE mAbs to 293F cells expressing different huRAGE deletion mutants. While initial epitope mapping indicated that L37 is a V domain binder, subsequent experiments suggest that it may actually be interacting with the C1/C2 domain of RAGE.
  • Several anti-huRAGE mAbs were shown to inhibit
  • IFNa gene signature PBMCs from healthy donors were stimulated for 4h with 50% sera from SLE patients. Total RNA was purified and expression of type I IFN- inducible genes, including DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, IFI35 were measured by real-time QRT-PCR analysis.
  • IFNa-induced gene expression The antibodies were also tested for their ability to inhibit the IFN ⁇ gene signature profile. As follows, PBMCs from healthy donors were stimulated for 4h with 50% sera from SLE patients in the presence of anti- huRAGE antibodies or an unrelated control antibody at a concentration of 10 ⁇ g/mL. In addition a RAGE-Fc fusion molecule was used as a positive control. The inhibition values are normalized to the negative control Ab. Following incubation the expression profile was tested as described above. The representative data are shown in Figures 5A-B and the averaged results from three repeats are summarized in Table 2. Specific results shown in Figure 6 demonstrate that the L30 (LpxR30#12) antibody inhibits the IFN- ⁇ gene signature induced by anti-dsDNA+ SLE serum.
  • SLE serum can stimulate IFN ⁇ gene signature (up-regulation of IFN inducible genes) in PBMCs.
  • This system can be used to examine the ability of therapeutic agents to modulate the IFN ⁇ gene signature, i.e., reduce the up-regulation of IFN inducible genes.
  • This system was used to examine the anti-huRAGE antibodies described above (see Example 1). The data summarized in Table 2 and detailed in Table 5 indicate that several anti-RAGE mAbs inhibited the IFN ⁇ gene signature by - 45-50 % which was comparable to the inhibition by soluble RAGE-Fc (54 ⁇ 11%). Interestingly, these mAbs also inhibit the binding between RAGE and HMGBl complexed with CpG DNA.
  • a RAGE domains expressed on 293 cells.
  • b 1% SLE serum + 5% MEF lysate stimulate huPBMC, measure IFNa secretion.
  • d ELISA using plate-bound RAGE-Fc and HMGB 1/CpG complex, detection by anti-HMGBl (S16).
  • e cell surface staining of huPBMC pre-incubated with 50% SLE serum for 4 hours.
  • f sequence analysis showed that these two clones (L4 and L30) were identical.
  • Bovine RAGE (Ace. No. NMJ 73982) 11 V H nucleotide sequence of S2 4 V L amino acid sequence of S2 12 V L nucleotide sequence of L30 5 V H amino acid sequence of S2 13 V H nucleotide sequence of L30 6 V L amino acid sequence of L30 14 V L nucleotide sequence of L37
  • aEC50 values determined against huRAGE-His by ELISA except as noted in .
  • bEC50 value determined against huRAGE-Fc by ELISA cValues determined against huRAGE-Fc by surface plasmon resonance.

Abstract

Antibodies that bind RAGE and inhibit the binding between RAGE and HMGBl complexed with CpG DNA are disclosed. The antibodies of the present invention and pharmaceutical compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range conditions characterized by the presence of DNA immune complexes and/or associated with the expression of one or more type-I IFN- inducible gene.

Description

Anti-RAGE Antibodies and Methods of Use Thereof
1. BACKGROUND OF THE INVENTION
[0001] The pattern recognition receptor, RAGE (receptor for advanced glycation end products) is a multi-ligand type I transmembrane glycoprotein of the immunoglobulin superfamily. RAGE ligands include advanced glycation end products (AGE), amyloid-β (A- beta; Aβ) peptide, proinflammatory cytokine-like mediators of the SlOO/calgranulin family, and high mobility group box 1 protein (HMGBl). RAGE has been implicated in several inflammatory disorders and diabetes, and recently we reported that HMGBl and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis (see, e.g., Tian et al., 2007, Nat. Immunol. 8:487-495). Furthermore, increased IFNα levels are associated with systemic lupus erythematosus (SLE) and are believed to play an important role in disease pathology. RAGE has also been shown to have an active, pathogenic role in a wide range of amyloidogenic diseases and disorders, including Alzheimer's disease.
[0002] A number of significant human disorders are associated with an increased production of ligands for RAGE or with increased production of RAGE itself. Consistently effective therapeutics are not available for many of these disorders. These disorders include, for example, many chronic inflammatory diseases, including rheumatoid and psoriatic arthritis, SLE, chronic obstructive pulmonary disease (COPD), scleroderma, and intestinal bowel disease, cancers, diabetes and diabetic nephropathy, amyloidoses, Alzheimer's disease, cardiovascular diseases, and sepsis. It would be beneficial to have safe and effective treatments for such RAGE-related diseases and disorders.
2. SUMMARY OF THE INVENTION
[0003] The invention is based in part on the discovery of antibodies that bind RAGE and inhibit the binding between RAGE and HMGBl complexed with CpG DNA. The anti- RAGE antibodies of the present invention (also referred to herein as "antibodies of the invention") were also seen to inhibit type I IFN-inducible gene expression associated with systemic lupus erythematosus (SLE).
[0004] Accordingly, the present invention provides methods of inhibiting the interaction of RAGE and HMGBl and/or HMGB 1/CpG complexes with RAGE by administering the anti-RAGE antibodies of the instant invention. The present invention also provides methods of inhibiting type I IFN-inducible gene expression associated with SLE. [0005] The invention also includes methods for preventing or treating a RAGE- related disease or disorder in a subject having such a disease or disorder, that comprises administering to the subject a therapeutically effective amount of an anti-RAGE antibody or a RAGE-binding fragment thereof of the invention. [0006] The present invention provides methods of treating a subject having a disease or disorder characterized by amyloid deposit of Aβ by administering a therapeutically effective amount of an antibody that binds specifically to RAGE (i.e., anti-RAGE antibodies) and inhibits the binding of a RAGE ligand (e.g., HMGB, Aβ), also referred to herein as a "binding partner". The diseases or disorders treatable by the disclosed methods may be characterized by amyloid deposit of Aβ in brain, such as occurs in Alzheimer's disease. Anti- RAGE antibodies as described herein may also be used to inhibit or reduce accumulation of amyloid deposit of Aβ in a subject, to inhibit or reduce neurodegeneration in a subject, to inhibit or reduce cognitive decline in a subject, and/or to improve cognition in a subject. The invention includes a method for preventing or treating a RAGE-related disease or disorder selected from the group consisting of sepsis, septic shock, including disorders or conditions such as community-acquired pneumonia, which result in sepsis or septic shock, listeriosis, inflammatory diseases, scleroderma, cancers, arthritis, rheumatoid arthritis, peritonitis, Crohn's disease, lupus, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury (SCI), allograft rejection, chronic acute inflammatory diseases, cardiovascular diseases, erectile dysfunction, diabetes, complications of diabetes, vasculitis, nephropathies, retinopathies, neuropathies, amyloidoses, preclinical Alzheimer's disease, Alzheimer's disease, and COPD. Such a method of the invention can comprise administering a composition comprising an anti-RAGE antibody or RAGE-binding fragment thereof of the invention in combination with one or more agents useful in the treatment of the RAGE-related disease or disorder that is to be treated. Such agents of the invention include antibiotics, anti-inflammatory agents, antioxidants, β-blockers, antiplatelet agents, ACE inhibitors, lipid-lowering agents, anti-angiogenic agents, and chemotherapeutics. [0007] In addition, the antibodies of the invention are useful for diagnostic applications.
3. BRIEF DESCRIPTION OF THE FIGURES
[0008] Figure 1. Alignment of the amino acid sequences of human, bovine (Neeper et ah, 1992), and rat (Renard et al, 1997) RAGE. Potential N-glycosylation sites are shown by bold underlining, and tyrosine residues (Y) are shown by light underlining. Cysteine residues involved in Ig domains are marked by asterisks. Boxed amino acids correspond to residues different among the three RAGE molecules.
[0009] Figure 2. Schematic diagram of human RAGE (huRAGE) depicting the domains and structure of RAGE and the various huRAGE deletion constructs used in the studies described in the Examples.
[0010] Figure 3. Example of reactive epitope mapping of anti-huRAGE antibody clone SNSR2.62#5 showing binding of this antibody clone to the Cl region.
[0011] Figure 4. Example of Inhibition of HMGB-1/CpG binding to immobilized huRAGE-Fc by N7 (SNSR7#2) and N262 (SNSR2.62#5). An unrelated mouse IgG antibody was used as a control.
[0012] Figure 5. Profiles of the effect of anti-huRAGE mAbs on the relative gene expression of IFITl, G1P2 (Panel A, top and bottom, respectively), MXl and RSAD2 (Panel B, top and bottom, respectively) in human PBMCs stimulated with sera from SLE patients. [0013] Figure 6 shows bar graphs depicting the relative expression of several IFN-α inducible genes (top row from left to right: IFITl, MXl, and DDX58; bottom row from left to right G1P2, OAS3, and RSAD2) by healthy human donor PBMCs following stimulation by 50% sera from SLE patients alone ("SLE") or in the presence of either the anti-RAGE Ab LpxR30#12 ("SLE / L30") or an unrelated control antibody ("SLE / Cont IgG"). [0014] Figure 7. The amino acid sequences and nucleotides encoding the variable regions of the light chains (VL) and the heavy (VH) of the mouse anti-RAGE antibodies of the invention. Underlined: CDRs (Kabat definition). A) S2 VL (SEQ ID NO: 4 and 10) and VH (SEQ ID NO:5 and 11); B) L30 VL (SEQ ID NO:6 and 12) and VH (SEQ ID NO:7 and 13); C) L37 VL (SEQ ID NO:8 and 14) and VH (SEQ ID NO:9 and 15). See Table 4 for legend for sequence listing.
4. DETAILED DESCRIPTION 4.1 Anti-RAGE Antibodies of the Invention
[0015] The invention is based in part on the discovery of antibodies that bind to the Receptor for Advanced Glycation End Products (RAGE) and inhibit the binding between RAGE and HMGBl alone or complexed with CpG DNA (such complexes are also referred to herein as "HMGB 1/CpG"). The HMGB (High Mobility Group Box) proteins are abundant chromatin-binding proteins located mainly in the nucleus where they regulate many transcriptional events by increasing the affinity of transcription factor-DNA interactions. There are three HMGB proteins (HMGBl, HMGB2 and HMGB3) that are highly conserved and consist of an A-box and a B-box with a carboxy-terminal tail. HMGBl is released from cells by two distinct mechanisms; it is passively released by necrotic cells and is also actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of the molecule (See, PCT/IB2003/005718). HMGB 1 has been implicated in inflammatory disorders such as sepsis and rheumatoid arthritis (see, for example International Patent Publications WO/2007/001422; WO 2007/084253 and WO 2007/076200). CpG belongs to a family of molecules referred to as "PAMPS." PAMPS are molecules which have structures called pathogen-associated molecular patterns. Non-limiting examples of PAMPs are, lipopolysaccharide (LPS) from the gram-negative cell wall, peptidoglycan, lipoteichoic acids from the gram-positive cell wall, flagellin, pilin, mannose rich glycans, bacterial and viral nucleic acids, N-formylmethionine found in bacterial proteins, double-stranded RNA from viruses, phosphorylcholine and other lipids common to microbial membranes and glucans such as lipoteichoic acids, glycolipids, and zymosan from fungal cell walls. HMGB can form an immunostimulatory complex with PAMPs that augments PAMP signaling. Accordingly, in one embodiment, antibodies of the invention may inhibit the binding of RAGE to HMGBl and/or RAGE binding to HMGB 1 complexed with a PAMP (such complexes are also referred to herein as "HMGB 1/P AMP"). In a specific embodiment, antibodies of the invention inhibit RAGE binding to HMGBl complexed CpG. In another specific embodiment, antibodies of the invention inhibit RAGE binding to HMGBl complexed to human DNA (such complexes are also referred to herein as "HMGB1/DNA"). In still another specific embodiment, an antibody of the invention inhibits RAGE binding to HMGBl associated with an immune complex (such complexes are also referred to herein as "HMGB 1/IC"). Immune complexes include but are not limited to those comprising cellular DNA and auto-immune antibodies such as anti-DNA antibodies.
[0016] In certain embodiments, the antibodies of the invention inhibit RAGE binding to one or more HMGBl complexes including, but not limited to, HMGB 1/P AMP, HMGB1/DNA and HMGB1/IC complexes. In other embodiments, the antibodies of the invention inhibit RAGE binding to one or more HMGBl complexes (e.g., HMGB 1/CpG complexes) by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%.
[0017] RAGE is known to bind numerous ligands in addition to HMGB 1. Non- limiting examples of RAGE ligands include advanced glycation end products (AGEs), adducts formed by glycation and/or glycoxidation (Baynes J.W., 1991, Diabetes. 1991, 40:405-412; Ahmed K.A., 2007, J Clin Biochem Nutr. 41 (2):97-105) that accumulate especially in disorders such as diabetes and renal failure; members of the SlOO/calgranulin family (e.g., calgranulin C (also known as ENRAGE and S100A12), SlOOAl, S100A4, SlOOAl 1, S100A13, SlOOB, and SlOOP); amyloid-β-peptide (Aβ); leukocyte integrins (e.g., Mac-1). In certain embodiments, the antibodies of the invention do not inhibit the binding of one or more RAGE ligand selected from the group consisting of: AGEs, S100A12, SlOOAl, S100A4, SlOOAl 1, S100A13, SlOOB, SlOOP, amyloid-β-peptide and Mac-1. In still other embodiments, the antibodies of the invention also inhibit the binding of one or more RAGE ligand selected from the group consisting of: AGEs, S100A12, SlOOAl, S100A4, SlOOAl 1, SlOOAl 3, SlOOB, SlOOP, amyloid-β-peptide and Mac-1. In certain embodiments, the antibodies of the invention selectively inhibit the binding of a subset of RAGE ligands including, but not limited to AGEs, S100A12, SlOOAl, S100A4, SlOOAI l, S100A13, SlOOB, SlOOP, amyloid-β-peptide and Mac-1. Non-limiting examples of such selectively would include antibodies that inhibit the binding of RAGE to HMGBl (alone or complexed) that also inhibit the binding of AGEs and/or Aβ but do not inhibit the binding of one or more SlOO family members, or antibodies that inhibit inhibit the binding of RAGE to HMGBl (alone or complexed) that also inhibit the binding of one or more SlOO family members but do not inhibit the binding of AGEs and/or Aβ. As described infra different RAGE ligands are known to be associated with different diseases and conditions, accordingly, one of skill in the art would recognize which RAGE ligand interactions to target for inhibition with the antibodies of the invention. In a specific embodiment, the antibodies of the invention inhibit the binding of RAGE to HMGBl (alone or complexed) and do not inhibit the binding of RAGE to human RAGE SlOOAl 2. In another specific embodiment, the antibodies of the invention inhibit the binding of RAGE to HMGBl (alone or complexed) and do not inhibit the binding of RAGE to bovine SlOOB.
[0018] In certain embodiments, the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR. In specific embodiments, the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%. In other specific embodiments, the antibodies of the invention inhibit the intracellular or extracellular localization of a RAGE molecule with a TLR selected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRI l.
[0019] In one embodiment, antibodies of the invention bind to RAGE and antigenic fragments thereof. In a specific embodiment, antibodies of the invention bind human RAGE and antigenic fragments thereof. Human RAGE is exemplified by GenBank™ accession number BC020669 (SEQ ID NO:1). As shown in Figure 1, RAGE is highly conserved in a number of species. Accordingly, the antibodies of the invention will bind epitopes conserved across species. In another embodiment, antibodies of the invention bind murine RAGE and antigenic fragments thereof. Murine RAGE is exemplified by GenBank™ accession number NM 007425. In another embodiment, antibodies of the invention bind rat RAGE and antigenic fragments thereof. Rat RAGE is exemplified by GenBank™ accession number NP 445788 (SEQ ID NO:2) and in Figure 1. In another embodiment, antibodies of the invention bind bovine RAGE and antigenic fragments thereof. Bovine RAGE is exemplified GenBank™ accession number NM 173982 (SEQ ID NO:3) and in Figure 1. Numerous splice variants and iso forms of RAGE have been identified (see for example GenBank™ accession numbers NM OOl 136, XM OOl 163454, DQ104254 and U.S. Patent Publication 2007008740) and one of skill in the art will recognize that antibodies of the invention may bind to one or more RAGE variants and or isoforms. In a specific embodiment, antibodies of the invention bind to RAGE and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human.
[0020] Human RAGE amino acid sequence (encoded by GenBank™ accession number BC02066)
[0021] MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPP QRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLP AVGIQDEGIFRCQAM NRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYP AGTLSWHL DGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRH RALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKD GVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVG GSGLGTLALALGILGGLGTAALLIGVILWQRRQRRGEERKAPENQEEEEERAELNQS EEPEAGESSTGGP (SEQ ID NO: 1)
[0022] In one embodiment, the antibodies of the invention bind a polypeptide comprising, or alternatively consisting of (or consisting essentially of) a RAGE polypeptide and antigenic fragments thereof, having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human RAGE polypeptide encoded by GenBank™ accession number BC020669.
[0023] The term RAGE "fragment" described herein includes a RAGE peptide or polypeptide comprising, or alternatively consisting of (or consisting essentially of) an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, at least contiguous 250 amino acid residues, at least contiguous 275 amino acid residues, at least contiguous 300 amino acid residues, at least contiguous 325 amino acid residues, at least contiguous 350 amino acid residues, at least contiguous 375 amino acid residues, or at least contiguous 400 amino acid residues, of the amino acid sequence of RAGE.
[0024] In certain embodiments, antibodies of the invention bind to a soluble RAGE fragment including, but not limited to, amino acids 24-336 of SEQ ID NO: 1 and fragments thereof. In certain embodiments, antibodies of the invention bind to a RAGE domain including, but not limited to, the V domain (amino acids 24- 129 of SEQ ID NO : 1 ), the C 1 domain (amino acids 130-234 of SEQ ID NO:1), the C2 domain (amino acids 235-336 of SEQ ID NO:1), the VCl domains (amino acids 24-234 of SEQ ID NO:1), and the C1C2 domain (amino acids 130-336 of SEQ ID NO:1) as disclosed by Xie et al., (2007, J. Biol. Chem., Vol. 282:4218-4231). In a specific embodiment, antibodies of the invention bind to the C2 domain (amino acids 235-336 of SEQ ID NO: 1) of RAGE as disclosed by Xie et al, supra. In other embodiments, antibodies of the invention bind to a RAGE domain including, but not limited to, the V domain (amino acids 31-106 of SEQ ID NO:1), the Cl domain (amino acids 137-214 of SEQ ID NO:1), the C2 domain (amino acids 252-308 of SEQ ID NO:1), the VCl domains (amino acids 31-214 of SEQ ID NO:1), and the C1C2 domain (amino acids 137-308 of SEQ ID NO: 1) as disclosed by Renard et al. (2007, The J. Pharm. Exp. Therap. Vol. 290:1458-1). In a specific embodiment, antibodies of the invention bind to the C2 domain (amino acids 252-308 of SEQ ID NO: 1) of RAGE as disclosed by Renard et al., supra. In still other embodiments, antibodies of the invention bind to a polypeptide comprising a RAGE domain including, but not limited to: the V domain, wherein said polypeptide comprises amino acids 1-121 of SEQ ID NO:1; the Cl domain, wherein said polypeptide comprises amino acids 122-233 of SEQ ID NO:1; the C2 domain, wherein said polypeptide comprises amino acids 234-342 of SEQ ID NO:1; the VCl domains, wherein said polypeptide comprises amino acids 1-233 of SEQ ID NO:1; and the C1C2 domain, wherein said polypeptide comprises amino acids 122-342 of SEQ ID NO:1, as disclosed herein (see, Section 6.1). In a specific embodiment, antibodies of the invention bind to a polypeptide comprising the C2 domain of RAGE, wherein said polypeptide comprises amino acids 234-342 of SEQ ID NO:1 as disclosed herein (see, Section 6.1).
[0025] Additional embodiments of the present invention are directed to antibodies of the invention that bind to a RAGE polypeptide comprising (or entirely consisting of) amino acids amino acids x to y of SEQ ID NO:1, where x represents an integer from 23 to 335, and where y represents an integer from 30 to 344.
[0026] In certain embodiments, antibodies of the invention bind to a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human RAGE C2 domain (amino acids 235-336 of SEQ ID NO:1) and antigenic fragments thereof. In a specific embodiment, the antibodies of the invention bind to a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to amino acids 234-342 of SEQ ID NO:1.
[0027] It will be understood by one of skill in the art that the percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined, for example, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A specific, non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al., Nucleic Acids Res., 29:2994-3005 (2001). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, as available on April 10, 2002. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1. [0028] Another, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl. Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988).
[0029] In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.accelrys.com, as available on August 31, 2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.cgc.com), using a gap weight of 50 and a length weight of 3.
[0030] It is understood by one of skill in the art that antibodies may discriminate between the same polypeptide isolated from different sources. Without wishing to be bound by any particular theory, a polypeptide of similar or identical amino acid sequence isolated from different sources may be distinguished by a number of differences including but not limited to, posttranslational modifications (e.g., phosphorylation, acetylation, methylation, glycosylation, etc.), alterations in overall structure (e.g., changes in disufide bonding and/or folding) and differences in any other molecules that the polypeptide may be associated with (e.g., salts, additional subunits such as polynucleotides and/or other polypeptides). In one embodiment, antibodies of the invention bind RAGE independently of posttranslational modifications and/or alterations in overall structure. In another embodiment, RAGE binding of the antibodies of the invention is dependent on posttranslational modifications and/or alterations in overall structure.
[0031] In one embodiment, antibodies of the invention inhibit the expression of one or more type I Interferon (IFN) genes or one or more type I IFN-inducible genes including but not limited to, DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35. In specific embodiments, antibodies of the invention inhibit the expression of one or more Type I interferon genes, or one or more type I IFN-inducible genes by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 120%, by at least 140%, by at least 160%, by at least
180%, or by at least 200%. In particular embodiments, antibodies of the invention inhibit the expression of one or more type I IFN genes by at least 25%. In particular embodiments, antibodies of the invention inhibit the expression of one or more type I IFN-inducible genes by at least 25%. [0032] The biochemical characteristics of the antibodies of the invention include, but are not limited to, isoelectric point (pi) and melting temperature (Tm). The binding characteristics of the antibodies of the invention include, but are not limited to, binding specificity, dissociation constant (Kd), epitope, ability to distinguish between various forms and/or preparations of RAGE (e.g., recombinant, native, acetylated) and ability to bind soluble and/or immobilized antigen. The functional characteristics of the antibodies of the present invention include, but are not limited to, inhibition of RAGE binding to HMGB- 1/CpG, inhibition of the intracellular or extracellular localization of a RAGE molecule with a TLR, and protection in one or more RAGE-related disease or disorder (e.g., sepsis, arthritis, Alzheimer's disease). [0033] In one embodiment, antibodies of the invention bind RAGE and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10"5 M, or of less than 10"6 M, or of less than 10"7 M, or of less than 10"8 M, or of less than 10~9 M, or of less than 10"10 M, or of less than 10"11 M, or of less than 10"12 M, or of less than 10~13 M, or of less than 5 xlO~13M, or of less than 10"14M, less than 5xlO"14M, or of less than 10"15M, or of less than 5X 10 15M. In still another embodiment, an antibody of the invention that binds RAGE and antigenic fragments thereof has a dissociation constant or IQ (koff/kon) of between about 10"7M and about 10"8M, between about 10"8M and about 10"9M, between about 10"9M and about 10"10M, between about 10"10M and about 10"11M, between about 10"11M and about 10"12M, between about 10"12M and about 10 13M, between about 10"13M and about 10'14M. In still another embodiment, an antibody of the invention that binds RAGE and antigenic fragments thereof has a dissociation constant or KJ (koff/kon) of between 10 M and 10 M, between 10"8M and 10"9M, between 10"9M and 10"10M, between 10"10M and 10"11M, between 10"11M and 10"12M, between 10"12M and 10"13M, between 10"13M and 10"14M. [0034] It is well known in the art that the equilibrium dissociation constant (KD) is defined as KfJk0n- (also commonly referred to as kd/ka) It is generally understood that a binding molecule {e.g., and antibody) with a low KD {i.e., high affinity) is preferable to a binding molecule {e.g., and antibody) with a high KD (i.e., low affinity). However, in some instances the value of the kon or £o#may be more relevant than the value of the KD. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. In certain embodiments, the antibodies of the invention have a lower KD for one antigen than for others.
[0035] The binding characteristics of the antibodies of the invention can be determined by methods known to those of skill in the art, for example, by use of surface plasmon resonance measurements, and as described herein in the Examples.
[0036] In another embodiment, antibodies of the invention bind RAGE and antigenic fragments thereof with a Koff rate of less than 10~3 s"1, less than 5x10~3 s"1, less than 10~4 s"1, less than 5xlO~4 s"1, less than 10~5 s"1, less than 5xlO~5 s"1, less than 10~6 s"1, less than 5xlO"6 s"1, less than 1(T7 s"1, less than 5xlO"7 s"1, less than 10"8 s"1, less than 5xlO"8 s"1, less than 10"9 s"1, less than 5x109 s"1, or less than 10"10 s"1.
[0037] In still another embodiment, antibodies of the invention bind RAGE and antigenic fragments thereof with a kon rate of at least 105 M-1S"1, at least 5 x 105 M-1S"1, at least 106M-1S"1, at least 5 x 106M-1S"1, at least 107M-1S"1, at least 5 x 107M-1S"1, or at least 108M"V \ or at least 109 MV.
[0038] In still another embodiment, antibodies of the invention bind RAGE and antigenic fragments thereof with an equilibrium dissociation constant or KD (koff/kon) of less than 10"5 M, or of less than 10"6 M, or of less than 10"7 M, or of less than 10"8 M, or of less than 10"9 M, or of less than 10~10 M, or of less than 10"11 M, or of less than 10~12 M, or of less than 10"13 M.
[0039] In certain embodiments, antibodies of the invention include particular antibodies (and antigen binding fragments and derivatives thereof) that specifically bind RAGE and antigenic fragments thereof. In particular are the antibodies referred to herein as "S2/1.40#9" which is abbreviated herein as "S 14"; "S2/1.50#2" which is abbreviated herein as "S15"; "S2/2.2#l" which is abbreviated herein as "S22"; "S2/2.13#5" which is abbreviated herein as "S21"; "S2/3.51#5" which is abbreviated herein as "S35"; "S2/4.146#18" which is abbreviated herein as "S41"; "S2/4.17#4" which is abbreviated herein as "S2"; "S2/6.23#2" which is abbreviated herein as "S62"; "SNSR7#2" which is abbreviated herein as "N7";
"SNSR2.5" which is abbreviated herein as "N25"; "SNSR2.62#5" which is abbreviated herein as "N262"; "SNSR2.63#2" which is abbreviated herein as "N263"; "LpxR4.12#2" which is abbreviated herein as "L4"; "LpxR14#l" which is abbreviated herein as "L 14"; "LpxR30#12" which is abbreviated herein as "L30"; "LpxR37#2" which is abbreviated herein as "L37"; "LpxR62#l" which is abbreviated herein as "L62"; "LpxR5.11" " which is abbreviated herein as "L5"; "F4/310#3" which is abbreviated herein as "F 10"; "F4/310#9" which is abbreviated herein as "F9"; and "F4/311#9" which is abbreviated herein as "Fl 1". Antibodies having at least one, at least two, at least three, at least four, at least five, or all six of the CDRs of these antibodies are specific embodiments of the invention. Isolated polynucleotides that encode these antibodies (and antigen binding fragments thereof) are also embodiments of the invention. Antibodies that bind to the same epitopes as these antibodies are also embodiments of the invention, as are antibodies that compete for binding with any of the above listed antibodies. The binding and functional characteristics for S 14, S 15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and FI l are listed in Table 2, 5, and 6. The nucleotide and cooresponding amino acid sequences of the H and L variable domains of the mouse anti-RAGE antibodies S2, L30, and L37 are shown in FIG. 7, panels A to C, respectively.
[0040] The hybridoma cell lines producing S2; L4; L30 and L37 were deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110- 2209) on June 26, 2007 and assigned ATCC Deposit Nos. PTA-8510, PTA-8509, PTA-8511 and PTA-8508, respectively. Subsequent to making the deposits, sequencing analysis revealed that L4 and L30 are independent isolates of the same clone. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Since the strains referred to are being maintained under the terms of the Budapest Treaty, they will be made available to a patent office signatory to the Budapest Treaty.
[0041] The present invention also encompasses variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) comprising one or more amino acid residue substitutions, additions and/or deletions in the variable light (VL) domain and/or variable heavy (VH) domain. The present invention also encompasses variants of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) with one or more additional amino acid residue substitutions, additions and/or deletions in one or more VL CDRS and/or one or more
VH CDRS. The antibody generated by introducing substitutions, additions and/or deletions in the VH domain, VH CDRS, VL domain and/or VL CDRS of the anti-RAGE antibodies of the invention (e.g., S14, S15, S22, S21, S35, S41, S2, S62, N7, N25, N262, N263, L4, L14, L30, L37, L62, L5, FlO, F9 and Fl 1) can be tested in vitro and in vivo, for example, for its ability to bind to a RAGE polypeptide (by, e.g., immunoassays including, but not limited to ELISA (Enzyme-Linked Immunosorbent Assay) and BIAcore), or for its ability to inhibit one or more RAGE activity.
[0042] It will be understood that the complementarity determining regions (CDRs) residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). Specifically, residues 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35
(CDRl), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al., supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
[0043] In other embodiments, the invention includes antibodies having at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs of an antibody disclosed herein. In a specific embodiment, an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has the three CDRs of the light chain variable region of any one of S2, L4, L30 and L37. In another specific embodiment, an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the three CDRs of the heavy chain variable region of any one of S2, L4, L30 and L37. In still another specific embodiment, an antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein both the light and the heavy chain variable regions have the three CDRs of the light and the heavy chain variable region, respectively, of any one of S2, L4, L30 and L37. [0044] Another embodiment of the present invention includes the introduction of conservative amino acid substitutions in any portion of an anti-RAGE antibody of interest, described infra (see Table 2). It is well known in the art that "conservative amino acid substitution" refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 1.
Table 1. Families of Conservative Amino Acid Substitutions
Family Amino Acids non-polar Trp, Phe, Met, Leu, He, VaI, Ala, Pro uncharged polar GIy, Ser, Thr, Asn, GIn, Tyr, Cys acidic/negatively charged Asp, GIu basic/positively charged Arg, Lys, His
Beta-branched Thr, VaI, He residues that influence chain orientation GIy, Pro
Aromatic Trp, Tyr, Phe, His
[0045] Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an anti-RAGE antibody of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions. In one embodiment, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions in the relative to the original anti-RAGE antibody. In another embodiment, the derivatives of an anti-RAGE antibody of the invention have conservative amino acid substitutions (e.g., supra) are made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to a RAGE polypeptide). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined. [0046] The term "conservative amino acid substitution" also refers to the use of amino acid analogs or variants. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," (1990, Science 247:1306-1310). [0047] Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab') fragments, disulfϊde-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above (e.g., RAGE binding antibody fragments). The terms "antibody" and "antibodies" as used herein specifically includes antigen binding antibody fragments. The antibodies of the present invention may be monospecific, bispecific, trispecifϊc or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al, J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al, J. Immunol. 148:1547-1553 (1992). [0048] Antibody-like and antibody-domain fusion proteins which bind RAGE are also contemplated as antibodies of the present invention. An antibody-like molecule is any molecule that has been generated with a desired binding property, see, e.g., PCT Publication Nos. WO 04/044011; WO 04/058821; WO 04/003019 and WO 03/002609. Antibody- domain fusion proteins may incorporate one or more antibody domains such as the Fc domain or the variable domain. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof. A large number of antibody- domain molecules are known in the art including, but not limited to, diabodies (dsFv)2 (Bera et al, 1998, J. MoI. Biol. 281:475-83); minibodies (homodimers of scFv-CH3 fusion proteins) (Pessi et al, 1993, Nature 362:367-9), tetravalent di-diabody (Lu et al, 2003 J.
Immunol. Methods 279:219-32), tetravalent bi-specific antibodies called Bs(scFv)4-IgG (Zuo et al, 2000, Protein Eng. 13:361-367). Fc domain fusions combine the Fc region of an immunoglobulin with a fusion partner which in general can be an protein, including, but not limited to, a ligand, an enzyme, the ligand portion of a receptor, an adhesion protein, or some other protein or domain. See, e.g., Chamow et al, 1996, Trends Biotechnol 14:52-60; Ashkenazi et al, 1997, Curr Opin Immunol 9:195-200; Heidaran et al, 1995, FASEB J. 9: 140-5. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; PCT
Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154:5590-5600; and ViI et al, 1992, Proc. Natl. Acad. Sci. USA 89:11337- 11341.
[0049] An antibody which can bind an intracellular epitope, (e.g., an intrabody) is useful for binding to and disrupting/inhibiting one or more activity of intracellular RAGE (e.g., nuclear and/or cytoplasmic RAGE). An intrabody comprises at least a portion of an antibody (e.g., an scFv) that is capable of specifically binding an antigen and which has been manipulated so that it can be expressed intracellularly. Generally, an intrabody does not contain sequences coding for its secretion. Such antibodies will bind antigen intracellularly. When combined with methods for expression and/or targeting to precise intracellular locations inside mammalian cells intrabodies are particularly useful for intracellular targets.
[0050] Generation of intrabodies is well-known to the skilled artisan and is described, for example, in U.S. Patent Nos. 6,004, 940; 6,072,036; 5,965,371. Further, the construction of intrabodies is discussed in Ohage and Steipe, 1999, J. MoI. Biol. 291 : 1119-1128; Ohage et al, 1999, J. MoI. Biol. 291 :1129-1134; and Wirtz and Steipe, 1999, Protein Science 8:2245- 2250; Stocks, M.R. Drug Disc. Today VoI 9, No. 22 November 2004. Recombinant molecular biological techniques may also be used in the generation of intrabodies.
[0051] In one embodiment, intrabodies of the invention retain at least about 75% of the binding effectiveness of the complete antibody (i.e., having the entire constant domain as well as the variable regions) to the antigen. In one embodiment, the intrabody retains at least 85%, at least 90%, or at least 95% of the binding effectiveness of the complete antibody.
[0052] Specific localization sequences can be attached to the intrabody polypeptide to direct the intrabody to a specific location. Intrabodies can be localized, for example, to the following intracellular locations: endoplasmic reticulum (Munro et al, 1987, Cell 48:899- 907; Hangejorden et al, 1991, J. Biol. Chem. 266:6015); nucleus (Lanford et al, 1986, Cell 46:575; Stanton et α/., 1986, PNAS 83:1772; Harlow et al, 1985, MoI. Cell Biol. 5:1605; Pap et al, 2002, Exp. Cell Res. 265:288-93); nucleolar region (Seomi et al, 1990, J. Virology 64:1803; Kubota et al, 1989, Biochem. Biophys. Res. Comm. 162:963; Siomi et al, 1998,
Cell 55:197); endosomal compartment (Bakke et al, 1990, Cell 63:707-716); mitochondrial matrix (Pugsley, A. P., 1989, "Protein Targeting", Academic Press, Inc.); Golgi apparatus (Tang et al, 1992, J. Bio. Chem. 267:10122- 6); liposomes (Letourneur et al, 1992, Cell 69:1183); peroxisome (Pap et al, 2002, Exp. Cell Res. 265:288-93); bans Golgi network (Pap et al, 2002, Exp. Cell Res. 265:288-93); and plasma membrane (Marchildon et al., 1984, PNAS 81 :7679-82; Henderson et al, 1987, PNAS 89:339-43; Rhee et al., 1987, J.
Virol.61:1045-53;Schultzetal, 1984, J. Virol. 133:431-7; Otsuyamaetal., 1985, Jpn. J. Can. Res. 76: 1132-5; Ratner et al, 1985, Nature 313:277- 84).
[0053] Recombinantly expressed intrabody may be administered to a patient to mediate a prophylactic or therapeutic effect. To direct the intrabody intracellularly the intrabody polypeptide is associated with a "membrane permeable sequence". Membrane permeable sequences are polypeptides capable of penetrating through the cell membrane from outside of the cell to the interior of the cell. When linked to another polypeptide, membrane I permeable sequences can also direct the translocation of that polypeptide across the cell membrane as well. Useful membrane permeable sequence include the hydrophobic; region of a signal peptide (see, e.g., Hawiger, 1999, Curr. Opin. Chem. Biol. 3:89-94;
Hawiger, 1997, Curr. Opin. Immunol. 9:189-94; U.S. Patent Nos. 5,807,746 and 6,043,339). The sequence of a membrane permeable sequence can be based on the hydrophobic region of any signal peptide. The signal peptides can be selected, e.g., from the SIGPEP database (see e.g., von; Heijne, 1987, Prot. Seq. Data Anal. 1:41- 2; von Heijne and Abrahmsen, 1989, FEBS Lett.; 224:439-46). When a specific cell type is to be targeted for insertion of an intrabody; polypeptide, the membrane permeable sequence is preferably based on a signal peptide endogenous to that cell type. In another embodiment, the membrane permeable sequence is a viral protein {e.g., Herpes Virus Protein VP22) or fragment thereof (see e.g., Phelan et al, 1998, Nat. Biotechnol. 16:440-3). A membrane permeable sequence with the appropriate properties for a particular intrabody and/or a particular target cell type can be determined empirically by assessing the ability of each membrane permeable sequence to direct the translocation of the intrabody across the cell membrane.
[0054] Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e., bispecifϊc antibodies, BsAbs), antibodies with additional specificities such as trispecifϊc antibodies are encompassed by the instant invention. Examples of BsAbs include without limitation those with one arm directed against a RAGE epitope and the other arm directed against any other antigen. Methods for making bispecifϊc antibodies are known in the art. Traditional production of full-length bispecifϊc antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature, 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, 1991, EMBO J., 10:3655-3659. A more directed approach is the generation of a Di-diabody a tetravalent bispecific antibody. Methods for producing a Di-diabody are known in the art (see e.g., Lu et al, 2003, J Immunol Methods 279:219-32; Marvin et al, 2005, Acta Pharmacolical Sinica 26:649).
[0055] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when, the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
[0056] In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm (e.g., a RAGE epitope such as the C2 domain), and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, 1986, Methods in Enzymology, 121 :210. According to another approach described in WO96/27011 , a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
[0057] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
[0058] Antibodies with more than two valencies incorporating at least one hinge modification of the invention are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
[0059] Other antibodies specifically contemplated are "oligoclonal" antibodies. As used herein, the term "oligoclonal" antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. Methods for generating oligoclonal antibodies are known in the art. See, e.g., "Examples Section", example 1, PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In certain embodiments, oligoclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., RAGE). Those skilled in the art will know or can determine what type of antibody or mixture of antibodies is applicable for an intended purpose and desired need. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to a RAGE antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-RAGE antibody). The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
[0060] The antibodies of the invention also encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. As outlined herein, the terms "antibody" and "antibodies" include the antibodies which bind RAGE described herein, full length antibodies and Fc variants thereof comprising Fc regions, or fragments thereof, comprising at least one novel amino acid residue described herein fused to an immunologically active fragment of an immunoglobulin or to other proteins as described herein. Such variant Fc fusions include but are not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) -Fc fusions, scFv-scFv-Fc fusions. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. [0061] Antibodies of the present invention also encompass antibodies that have half- lives (e.g., serum half-lives) in a mammal, (e.g., a human), of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half- lives of the antibodies of the present invention in a mammal, (e.g., a human), results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies having increased in vivo half- lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half- lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. 6,737056 and U.S. Patent Publication
No. 2003/0190311 and discussed in more detail below). [0062] In one embodiment, the antibodies of the invention may comprise modifications/substations and/or novel amino acids within their Fc domains such as, for example, those disclosed in Ghetie et al, 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al, 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al., 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al, 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925- 1933; Xu et al, 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571- 2575; Shields et al, 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al, 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Application Nos. 10/370,749 and PCT Publications WO 94/2935; WO 99/58572; WO 00/42072; WO 02/060919, WO
04/029207. Other modifications/substitutions of the Fc domain will be readily apparent to one skilled in the art.
[0063] Antibodies of the invention comprising modifications/substations and/or novel amino acid residues in their Fc regions can be generated by numerous methods well known to one skilled in the art. Non- limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the variable regions of an antibody of the invention may be subcloned into a vector encoding an Fc region comprising one or modifications/substations and/or novel amino acid residues. [0064] Antibodies of the invention may also be modified to alter glycosylation, again to alter one or more functional properties of the antibody. In one embodiment, the glycosylation of the antibodies of the invention is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861.
[0065] Additionally or alternatively, an antibody of the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Antibodies having reduced sialyation have also been shown to have increased ADCC activity while antibodies with increased sialylation have been shown to have reduced ADCC activity and anti-inflammatory activity (Scallon et al. (2006) MoI Immunol, 7:761-72 and Kaneko et al. (2006) Science, 313:670-3). Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.
Biotech. 17:176-1; Jassal et al. (2001) Biochem Biophys Res Comm, 286:243-249, as well as, European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342.
[0066] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
[0067] The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding a RAGE polypeptide or fragment thereof and/or generating a desired response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
[0068] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homo log of a polypeptide of the present invention are included. Antibodies that bind polypeptides (and polypeptide fragments) with at least 99%, at leat 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a human RAGE polypeptide (e.g., a human RAGE C2 domain) of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human RAGE proteins and the corresponding epitopes thereof.
[0069] Antibodies that do not bind any other analog, ortholog or homolog or RAGE, such as antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a RAGE polypeptide of the present invention are also included in the present invention.
[0070] In one embodiment, antibodies or antigen binding fragments thereof that specifically bind to a RAGE polypeptide or fragment thereof prevent/antagonize/inhibit one or more of the following: RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG), expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes, including but not limited to, DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, IFI35. These activities may be assayed by one or many known methods in the art. See, e.g., US20040005316, 6,468,533 and 6,448,223, and Section 6 entitled "Examples" infra.
[0071] The term "inhibitory concentration 50%" (abbreviated as "IC50") represents the concentration of an inhibitor (e.g., an antibody of the invention) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets (e.g., RAGE). It will be understood by one in the art that a lower IC50 value corresponds to a more potent inhibitor. [0072] In one embodiment, the antibodies of the invention inhibit the expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes by at least 30% with an IC50 of less than 5000 ng/ml, or of less than 4000 ng/ml, or of less than 3000 ng/ml, or of less than 2000 ng/ml, or of less than 1000 ng/ml, or of less than 500 ng/ml, or of less than 250 ng/ml, or of less than 100 ng/ml, or of less than 50 ng/ml, or of less than 10 ng/ml, or of less than 5 ng/ml. In another embodiment, the antibodies of the invention inhibit expression of one or more Type I IFN genes, or one or more type I IFN-inducible genes by at least 30% with an IC50 of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM.
[0073] In one embodiment, the antibodies of the invention inhibit RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG) with an IC50 of less than 5000 ng/ml, or of less than 4000 ng/ml, or of less than 3000 ng/ml, or of less than 2000 ng/ml, or of less than 1000 ng/ml, or of less than 500 ng/ml, or of less than 250 ng/ml, or of less than 100 ng/ml, or of less than 50 ng/ml, or of less than 10 ng/ml, or of less than 5 ng/ml. In another embodiment, the antibodies of the invention inhibit RAGE binding to HMGBl and/or RAGE binding to HMGBl complexed with a PAMP (e.g., LPS, CpG) with an IC50 of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM.
[0074] The term "effective concentration 50%" (abbreviated as "EC50") represents the molar concentration of an antibody of the invention that produces 50% of the maximum possible binding activity at the target molecule (e.g., RAGE). It will be understood by one of ordinary skill in the art that a lower EC50 value corresponds to a more potent binder. Assays to determine RAGE binding activity of the antibodies of the invention are known to those of ordinary skill in the art, including the ELISA binding assay for EC50 determination described herein in the Examples. In some embodiments, using an ELISA assay in which huRAGE-His is coated onto wells of microtiter plates, antibodies of the invention bind to RAGE with an EC50 value of less than 1000 nM, or of less than 500 nM, or of less than 250 nM, or of less than 100 nM, or of less than 50 nM, or of less than 25 nM, or of less than 10 nM, or of less than 5 nM, or of less than 0.25 nM, or of less than 0.1 nM, or of less than 0.01 nM, or of less than 0.001 nM. In particular embodiments, antibodies of the invention bind to RAGE with an EC50 value of less than 0.01 nM, or of less than 0.001 nM. [0075]
4.2 Methods of Generating and Screening Antibodies of the Invention
[0076] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a RAGE polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. [0077] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et aL, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et aL, in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N. Y., 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. [0078] A "monoclonal antibody" may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
[0079] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a RAGE polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
[0080] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention. [0081] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.
[0082] The antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et ah, J. Immunol. Methods 182:41-50 (1995); Ames et ah, J. Immunol. Methods 184:177-186 (1995); Kettleborough et ah, Eur. J. Immunol. 24:952-958 (1994); Persic et ah, Gene 187 9-18 (1997); Burton et ah, Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108. [0083] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6): 864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240:1041-1043 (1988). [0084] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).
[0085] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397. Humanized antibodies are antibody molecules from non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323 (1988)).
Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
[0086] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
[0087] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[0088] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/technology 12:899-903 (1988)).
[0089] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity. [0090] If the antibody is used therapeutically in in vivo applications, the antibody is preferably modified to make it less immunogenic in the individual. For example, if the individual is human the antibody is preferably "humanized"; where the complementarity determining region(s) of the antibody is transplanted into a human antibody (for example, as described in Jones et al, Nature 321 :522-525, 1986; and Tempest et al., Biotechnology 9:266-273, 1991).
[0091] Phage display technology can also be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-RAGE antibodies or from naive libraries (McCafferty et al., Nature 348:552-554, 1990; and Marks, et al, Biotechnology 10:779-783, 1992). The affinity of these antibodies can also be improved by chain shuffling (Clackson et al, Nature 352: 624-628, 1991).
[0092] The choice of polypeptide to be used for the generation can be readily determined by one skilled in the art. Polypeptides may be chosen such that the antibody generated will not significantly cross-react or specifically bind to another variant or isomer of the RAGE protein (see for example, Hudson B.I., et al., 1998, Diabetes, 47: 1155-1157; and U.S. Patent Publication 2007008740). Alternatively, polypeptides which share a large degree of homology between two or more variants of the RAGE protein may be used for the generation of an antibody that can specifically bind {i.e., cross-react) with multiple variants or isomers of the RAGE protein. In a specific embodiment, an antibody of the invention will bind cross-react or specifically bind to RAGE proteins from one or more different species, including but not limited to, human, mouse, primate, and monkey.
[0093] Antibodies or antigen binding fragments thereof that bind to a RAGE polypeptide can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
5.3 Polynucleotides Encoding Antibodies
[0094] The invention further provides polynucleotides comprising a nucleotide sequence encoding a high affinity antibody of the invention and antigen binding fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode an antibody that specifically binds to a RAGE polypeptide (e.g., human, murine, or rat RAGE or fragments thereof). In a particular embodiment, a polynucleotide of the invention encodes an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:1 or fragments thereof. In another embodiment, a polynucleotide of the invention encodes an antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a polynucleotide of the invention encodes an antibody that binds to a soluble RAGE fragment including, but not limited to, amino acids 24-336 of SEQ ID NO:1 and fragments thereof. In other embodiments, a polynucleotide of the invention encodes an antibody that binds to a RAGE domain including, but not limited to, the V domain (amino acids 24- 129 of SEQ ID NO : 1 ), the C 1 domain (amino acids 130-234 of SEQ ID NO:1), the C2 domain (amino acids 235-336 of SEQ ID NO:1), the VCl domains (amino acids 24-234 of SEQ ID NO:1), and the C1C2 domain (amino acids 130-336 of SEQ ID NO:1). In a specific embodiment, a polynucleotide of the invention encodes an antibody that binds to the C2 domain (amino acids 235-336 of SEQ ID NO: l) ofRAGE.
[0095] Nucleic acids of the invention also comprise a nucleotide sequence encoding any of the antibody variable region amino acid sequences shown in SEQ ID NOs:4, 5, 6, 7, 8, and 9. Specific nucleic acids of the invention include but are not limited to SEQ ID NOs: 10- 15. [0096] Nucleic acids of the invention may also comprise a nucleotide sequence encoding an anti-RAGE antibody variable region having an amino acid sequence that is substantially identical to any of the amino acid sequences shown in SEQ ID NOs:4-9, including a nucleotide sequence encoding an amino acid sequence that is at least 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to any of SEQ ID NOs:4-9. Nucleic acids of the invention may also comprise a nucleotide sequence encoding an anti-RAGE antibody variable region having an sequence that is substantially identical to any of the nucleotide sequences shown in SEQ ID NOs: 10- 15, including a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to any of SEQ ID NOs:10-15.
[0097] Sequences are compared for maximum correspondence using a sequence comparison algorithm using the full-length variable region encoding sequence of any one of SEQ ID NOs:4-9, a nucleotide sequence encoding a full length variable region having any one of the sequences shown in SEQ ID NOs:4-9, the nucleotide sequence of SEQ ID NOs : 10- 15 as the query sequence, as described herein, or by visual inspection.
[0098] Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population. An allelic difference may be as small as one base pair. Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations. A mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
[0099] Substantially identical nucleic acids are also identified as nucleic acids that hybridize specifically to or hybridize substantially to the full length of any nucleotide sequence encoding an antibody variable region amino acid sequence shown in SEQ ID
NOs:4-9, or to the the nucleotide sequence of SEQ ID NOs:10-15, under stringent conditions. In the context of nucleic acid hybridization, two nucleic acid sequences being compared may be designated a probe and a target. A probe is a reference nucleic acid molecule, and a target is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules. A target sequence is synonymous with a test sequence.
[0100] For hybridization studies, useful probes are complementary to or mimic at least about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention. Preferably, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any nucleotide sequence encoding an antibody variable region amino acid sequence shown in SEQ ID NOs:4-9, or the the nucleotide sequence of SEQ ID NOs: 10-15. Such fragments may be readily prepared, for example, by chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production. [0101] By "stringent hybridization conditions" is intended overnight incubation at 420C in a solution comprising: 50% formamide, 5X SSC (750 rnM NaCl, 75 niM trisodium cirate), 50 rnM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 650C.
[0102] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[0103] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
[0104] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions. [0105] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains of the antibodies of the invention may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability . As described supra, using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, J. MoI. Biol. 278 : 457-479 (1998) for a listing of human framework regions).
Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
[0106] Additionally or optionally, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art. [0107] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al, Proc. Natl. Acad. Sci. 81 :851-855 (1984); Neuberger et al, Nature 312:604- 608 (1984); Takeda et al, Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
[0108] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al, Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al, Science 242:1038-1041 (1988)). 5.4 Antibody Conjugates And Derivatives
[0109] Antibodies of the invention include derivatives that are modified (i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment). For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
[0110] Antibodies or antigen binding fragments thereof with increased in vivo half- lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
[0111] Further, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half- life in vivo. The techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413, 622. The present invention encompasses the use of antibodies or antigen binding fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. [0112] The present invention encompasses the use of antibodies or antigen binding fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al, 1994, Immunol. Lett. 39:91-99; U.S. Patent No. 5,474,981; Gillies et al, 1992, PNAS 89:1428-1432; and Fell et al, 1991, J. Immunol. 146:2446-2452.
[0113] The present invention further includes compositions comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154:5590-5600; and ViI et al, 1992, Proc. Natl. Acad. Sci. USA 89:11337- 11341.
[0114] Additional fusion proteins, e.g., of antibodies that specifically bind an antigen {e.g., supra), may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffiing (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activities of antibodies of the invention or antigen binding fragments thereof (e.g. , antibodies or antigen binding fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al, 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson, et al, 1999, J. MoI. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308- 313. Antibodies or antigen binding fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to an Antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
[0115] Moreover, the antibodies or antigen binding fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767) and the "flag" tag.
[0116] In other embodiments, antibodies of the present invention or analogs or derivatives thereof are conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (1311, 1251, 1231, 121I,), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, 111In,), and technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re,142 Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and U7Tin; positron emitting metals using various positron emission tomographies, noradioactive paramagnetic metal ions, and molecules that are radiolabeled or conjugated to specific radioisotopes.
[0117] The present invention further encompasses uses of antibodies of the invention or antigen binding fragments thereof conjugated to a therapeutic agent.
[0118] An antibody or antigen binding fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include ribonuclease, monomethylauristatin E and F, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine
(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT publications WO 03/075957.
[0119] Further, an antibody or antigen binding fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., 1994, J Immunol, 6:1567), and VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")), or a growth factor (e.g., growth hormone ("GH")). [0120] Moreover, an antibody can be conjugated to therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelator is l,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, CHn Cancer Res. 4:2483; Peterson et al, 1999, Bioconjug. Chem. 10:553; and Zimmerman et al, 1999, Nucl. Med. Biol. 26:943. [0121] Techniques for conjugating therapeutic moieties to antibodies are well known.
Moieties can be conjugated to antibodies by any method known in the art, including, but not limited to aldehyde/Schiff linkage, sulphydryl linkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage, enzymatically degradable linkage (see generally Garnett, 2002, Adv Drug Deliv Rev 53:171). Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62:119. [0122] Methods for fusing or conjugating antibodies to polypeptide moieties are known in the art. See, e.g., U.S. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995, J Immunol 154:5590; and ViI et al., 1992, PNAS USA 89:11337. The fusion of an antibody to a moiety does not necessarily need to be direct, but may occur through linker sequences. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, CHn Cancer Res 4:2483; Peterson et al, 1999, Bioconjug Chem 10:553; Zimmerman et al, 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.
[0123] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
[0124] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. [0125] The therapeutic moiety or drug conjugated to an Fc variant of the invention should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject. A clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to an Fc variant of the invention: the nature of the disease, the severity of the disease, and the condition of the subject.
4.3 Methods of Producing Antibodies
[0126] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
[0127] Recombinant expression of an antibody of the invention, or antigen binding fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
[0128] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[0129] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, 3T3, PerC6 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)). Also see, e.g., U.S. patents 5827739, 5879936, 5981216, and 5658759. [0130] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[0131] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
[0132] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome {e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts {e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81 :355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
[0133] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications {e.g., glycosylation) and processing {e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, NSO, Per.Cβ and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst. [0134] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
[0135] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, Cell 11 :223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Proc Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596
(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11 (5): 155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al, J. MoI. Biol. 150:1 (1981).
[0136] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., MoI. Cell. Biol. 3:257 (1983)).
[0137] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[0138] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or antigen binding fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. [0139] Moreover, the antibodies or antigen binding fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag. 4.4 Assays for Antibody Binding and Activity
[0140] The antibodies of the invention may be assayed for specific (i.e., immunospecific) binding by any method known in the art. The immunoassays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, cell surface binding assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[0141] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 40C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0142] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
[0143] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York at 11.2.1.
[0144] Cell surface binding assays comprise identifying or generating a cell which displays the antigen on its cell surface, adding the antibody of interest to the cell and incubating for a period of time, and detecting the presence of the antibody on the cells surface. Such detection generally employs methods such as fluorescence activated cell sorting (FACS) analysis. The antibodies to be tested may either directly coupled to a detectable compound such as a fluorescent dye, or alternative may be detected using a secondary antibody coupled to a detectable compound.
[0145] The binding affinity and other binding properties (e.g., off-rate of an antibody- antigen interaction) of an antibody to an antigen may be determined by a variety of in vitro assay methods well known in the art including for example, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.
[0146] The antibodies of the invention may be assayed for biological activity by any method known in the art, and as disclosed herein, see the Examples Section 6.2. [0147] The protocols and formulations of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific therapeutic protocol formulation or combination therapy of the invention is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise contacted with a formulation of the invention, and the effect of such a formulation upon the tissue sample is observed. The tissue sample can be obtained by biopsy from the patient. This test allows the identification of the therapeutically most effective prophylactic or therapeutic agent(s) for each individual patient. In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in an autoimmune disorder, an inflammatory disorder, a disorder mediated by RAGE and/or HMGBl, a disorder characterized by the presence of DNA immune complexes, a disorder TNF-alpha mediated inflammation, to determine if a formulation of the invention has a desired effect upon such cell types. For example, a lower level of expression of one or more type-I IFN -inducible gene and/or a lower level of proinflammatory cytokines produced by the contacted cells indicates that the composition of the invention may be effective to treat the disorder or condition in the patient. Alternatively, instead of culturing cells from a patient, a formulation of the invention may be screened using cells which can be stimulated to stimulate the expression of one or more type-I IFN -inducible gene such as for example peripheral blood mononuclear cells (PBMCs), THP-I cells or Macrophages (M0s). Many assays standard in the art can be used to assess IFN-inducible gene expression and/or cytokine production including ELISA assays, realtime PCR. Such methods are well known in the art and exemplified herein, see the Examples Section 6.2.
[0148] Prophylactic or therapeutic agents can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc.
[0149] Animal and cell culture models for Alzheimer's disease and sepsis are widely used and are known and described in the art, see for example, U.S. 6,717,031; U.S. 5,994,084; U.S. 20080038227; PCT publications WO 2003/093442 and WO2002/05634. [0150] Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the combinatorial therapies disclosed herein for treatment or prevention of RAGE-related and HMGBl -mediated disorders, diseases or conditions (e.g., sepsis, arthritis, Alzheimer's disease, COPD, scleroderma). [0151] 4.5 Antibodies, Antibody Compositions of the Invention and Therapeutic and/or Prophylactic Administration Thereof
[0152] The present invention encompasses anti-RAGE antibodies as disclosed herein and is also directed to antibody compositions referred to herein as "antibody compositions of the invention," "compositions of the invention" or more simply as "compositions". In certain embodiments, the compositions of the invention comprise one or more antibody of the invention in a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which an antibody of the invention may be combined and which, following the combination, can be used to administer the antibody of the invention to a subject. These antibody compositions are also referred to herein as "pharmaceutical compositions".
[0153] The terms "disease", "disorder" and "condition" are used interchangeably herein to refer to a physiological state that can be prevented or treated by administration of an antibody as described herein.
[0154] The present invention further includes methods for treating a disorder or condition characterized by TNF-alpha mediated inflammation and or conditions characterized by the presence of DNA immune complexes, including both acute and chronic inflammatory conditions, comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition of the invention. Chronic inflammatory conditions are characterized by an inflammatory response of prolonged duration - weeks, months, or even indefinitely which results in tissue damage that is often permanent. Acute inflammatory conditions are usually characterized by a sudden onset of symptoms including, increased vascular permeability, oedema, systemic fever often resulting in tissue necrosis and may result in death.
[0155] In certain embodiments, compositions of the invention comprise antibodies that bind to RAGE (e.g., an epitope within the polypeptide encoded by GenBank™ accession number BC020669). In another embodiment, compositions comprise antibodies of the invention that bind to the C2 domain of RAGE (e.g., an epitope within amino acids 234-342 or amino acids 235-336 of SEQ ID NO: 1).
[0156] Compositions of the invention can comprise the antibodies of the invention alone or in combination with other active therapeutic molecules and/or adjuvants such as steroids, other anti-inflammatory molecules, or other antibody therapeutics.
[0157] The present invention further provides methods of preventing, managing, treating or ameliorating a disorder, or one or more symptoms thereof, including, but not limited to, a disorder characterized by TNF-alpha mediated inflammation and/or by the presence of DNA immune complexes and/or an HMGBl -mediated disorder or condition, said methods comprising administering to a subject in need thereof an effective amount of an antibody composition of the invention. The methods also encompass the administration, to a subject in need thereof, an effective amount of an antibody composition of the invention in combination with an addition therapeutic agent. Any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of a disorder, characterized by TNF-alpha mediated inflammation and/or by the presence of DNA immune complexes and/or an HMGBl -mediated disorder or condition can be used in combination with an antibody composition of the invention. Examples of such agents include, but are not limited to, immunomodulatory agents, an anti- angiogenic agents, anti-inflammatory agents and TNFα antagonists.
[0158] In a specific embodiment, the present invention provides a method for treating an HMGBl -mediated disorder or condition, comprising administering an effective amount of an antibody composition of the invention. HMGBl -mediated disorders or conditions include, but are not limited to, systemic lupus erythematosus, inflammatory lupus nephritis psoriasis, Sjogren's disease, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, COPD, scleroderma, ulcerative colitis, inflammatory bowel disease, type I diabetes, allograft rejection and graft-versus-host disease.
[0159] In another specific embodiment, the present invention provides a method for treating a RAGE -mediated disorder or condition, comprising administering an effective amount of an antibody composition of the invention. RAGE -mediated conditions or disorders may be characterized generally as including any disorder in which an affected cell exhibits elevated expression of RAGE and/or one or more RAGE ligands. RAGE -mediated conditions or disorders may also be characterized as any disorder that is treatable (i.e., one or more symptoms may be eliminated or ameliorated) by a decrease in RAGE function. RAGE function can be decreased by administering an agent that disrupts the interaction between RAGE and one or more of its ligands. Increased expression of RAGE is associated with several pathological states, including diabetic vasculopathy, nephropathy, retinopathy, neuropathy, and other disorders, including Alzheimer's disease and immune/inflammatory reactions of blood vessel walls. RAGE ligands are produced in tissue affected with many inflammatory disorders, including arthritis (e.g., rheumatoid arthritis). In diabetic tissues, the production of RAGE is thought to be caused by the overproduction of advanced glycation end products (AGEs). This overproduction results in oxidative stress and endothelial cell dysfunction that leads to vascular disease in diabetics (WO 2004/016229; the entire teachings of which are incorporated herein by reference).
[0160] Deposition of amyloid in tissues causes a variety of toxic effects on cells and is characteristic of a number of diseases that may be termed amyloidoses. RAGE binds to beta-sheet fibrillar material, such as that found in amyloid-beta peptide, Abeta, amylin, serum amyloid A and prion-derived peptides. Increased expression of RAGE is also observed in tissues having amyloid structures. Accordingly, RAGE is involved in amyloid disorders. The RAGE-amyloid interaction is thought to result in oxidative stress leading to neuronal degeneration (WO 2004/016229). [0161] The present invention provides a method for treating a subject having a condition or disorder characterized by amyloid deposit of amyloid-β-peptide (Aβ), such as Alzheimer's disease, which comprises administering to a subject a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular Aβ.
[0162] The invention also provides a method of inhibiting or reducing accumulation of amyloid deposit of Aβ in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner. Also included within the invention is a method of inhibiting or reducing neurodegeneration in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner. The invention further includes a method of inhibiting or reducing cognitive decline, or improving cognition, in a subject, comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner. The invention also provides a method for treating a subject having an amyloidogenic disease or disorder characterized by amyloid deposit which comprises administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular Aβ.
[0163] The present invention provides a method for treating a subject having a condition or disorder characterized by amyloid deposit of Aβ, such as Alzheimer's disease, which comprises administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular Aβ, to a subject under conditions that generate a beneficial therapeutic response in the subject (e.g., reduction of plaque burden, inhibition of plaque formation, reduction of neuritic dystrophy, and improvement of cognitive function, e.g., rapidly improving cognition, and/or reversing, treating or preventing cognitive decline).
[0164] Diseases, disorders or conditions associated with amyloid deposits of Aβ in the brain include Alzheimer's disease, Down's syndrome and cognitive impairment. The latter can occur with or without other characteristics of an amyloidogenic disease. [0165] In addition to advanced glycation end products (AGEs), which form in prolonged hyperglycemic states, the ligands of RAGE include proteins having β-sheet fibrillar structures that are characteristic of amyloid deposits and pro-inflammatory mediators, including beta-amyloid protein (Aβ), serum amyloid (SAA) (fibrillar form),
SlOO/calgranulins (e.g., S100A12, SlOOB, S100A8-A9), and high mobility group box-1 chromosomal protein 1 (HMGBl, also known as amphoterin). There is growing awareness of the role of RAGE in the pathological progression of amyloidogenic diseases. In addition to its contributing to the pathogenesis of Alzheimer's disease, RAGE has been shown to be closely linked to cell stress and deposition of serum amyloid A (SAA) in spleen (Yan et al., 2000, Nature Med., 6:643-51). RAGE is associated with the accumulation of amyloid in kidneys and the tissue destruction leading to kidney failure of individuals with familial amyloidotic polyneuropathy (FAP) (Matsunaga et al., 2005, Scand. J. Clin. Lab. Invest.). The RAGE ligand amphoterin (HMGBl) also contains an amyloidogenic peptide, one that is highly homologous to the Alzheimer's Aβ peptide and forms amyloid-like peptides when released from the native protein (Kallijarvi et al., 2001, Biochem., 40:10032-7).
[0166] The interaction of Aβ with RAGE-bearing cells in the walls of blood vessels results in transport of Aβ across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-I), the latter mediating Aβ-induced vasoconstriction. Thus, the present invention also provides methods for reducing Aβ-induced vasoconstriction.
[0167] The inhibition of RAGE -ligand interaction has been shown to suppress the accumulation of Aβ in brain parenchyma in a transgenic mouse model for Alzheimer's-like disease (Deane et al., 2003, Nature Medicine 9:907-913). The active, pathogenic role of RAGE in a wide range of amyloidogenic diseases, disorders, and conditions makes it possible to provide therapeutic, beneficial treatment to patients with these amyloidogenic disorders by the method of the present invention, which provides antibodies that bind specifically to RAGE and inhibit the binding of a RAGE binding partner.
[0168] Patients amenable to treatment by the invention include individuals at risk of an Aβ-related disease or disorder or amyloidogenic disease or disorder but not showing symptoms, as well as patients presently showing symptoms. In the case of Alzheimer's disease, virtually anyone is at risk of suffering from Alzheimer's disease if he or she lives long enough. Therefore, the present methods can be administered prophylactically to the general population without the need for any assessment of the risk of the subject patient. [0169] The present methods are especially useful for individuals who are at risk for Alzheimer's disease, e.g., those who exhibit risk factors of Alzheimer's disease. Thus, the present methods are useful for preclinical Alzheimer's disease. The main risk factor for Alzheimer's disease is increased age. As the population ages, the frequency of Alzheimer's disease continues to increase. Current estimates indicate that up to 10% of the population over the age of 65 and up to 50% of the population over the age of 85 have Alzheimer's disease.
[0170] The methods of the invention can be used on both asymptomatic patients and those currently showing symptoms of disease. The antibodies used in such methods can be human, humanized, chimeric or nonhuman antibodies, or antigen binding fragments thereof (e.g., RAGE binding fragments), as described herein.
[0171] The present invention provides a method for inhibiting or reducing cognitive decline, and/or improving cognition, in a patient having or at risk for suffering from an Aβ- related disease, disorder or condition or amyloidogenic disease or disorder (e.g., Alzheimer's disease), comprising administering to the subject an effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner, in particular Aβ.
[0172] Several tests have been developed to assess cognitive skills or performance in human subjects, for example, subjects at risk for or having symptoms or pathology of dementia disorders (e.g., Alzheimer's disease). Cognitive deficits can be identified by impaired performance of these tests, and many treatments have been proposed based on their ability to improve performance in these tests. Although some tasks have evaluated behaviors or motor function of subjects, most tasks have been designed to test learning or memory.
[0173] Cognition in humans may be assessed using a wide variety of tests including, but not limited to, the following tests: ADAS-Cog (Alzheimer Disease Assessment Scale- Cognitive) (Rosen et al. (1984) Am J Psychiatry. 141(11):1356-64; IhI et al. (2000) Neuropsychobiol. 41(2): 102-7; and Weyer et al. (1997) Int Psychogeriatr. 9(2): 123-38); Blessed Test (Blessed et al. (1968) Br J Psychiatry 114(512):797-811); Cambridge Neuropsychological Test Automated Battery (CANTAB) (Swainson et al. (2001) Dement Geriatr Cogn Disord, 12:265-280; Fray & Robbins (1996) Neurotoxicol Teratol. 18(4):499- 504; and Robbins et al. (1994) Dementia 5(5):266-81; Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Clinical and Neuropsychological Tests include a verbal fluency test, Boston Naming Test, Mini Mental State Exam (MMSE) (Morris et al. (1988) Psychopharmacol Bull. 24(4):641-52; Morris et al. (1989) Neurology 39(9): 1159-65; and Welsh et al. (1991) Arch Neural. 48(3):278-81); Mini Mental State Exam (MMSE) (Folstein et al. (1975) J Psychiatr Res. 12:196-198; Cockrell & Folstein (1988) Psychopharm Bull. 24(4):689-692; and Crum et al. (1993) J. Am. Med. Association 18:2386-2391); Seven- Minute Screen (Solomon & Pendlebury (1998) Fam Med. 30(4):265-71, Solomon et al.
(1998) Arch Neural. 55(3):349-55). [0174] Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors well known in the art, including increased age and certain genetic predispositions. In addition, a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of cerebrospinal fluid (CSF) tau and Aβ42 levels. Elevated tau and decreased Aβ42 levels signify the presence of AD. Individuals suffering from Alzheimer's disease can also be diagnosed by Alzheimer's Disease and Related Disorders Association (ADRDA) criteria.
[0175] The anti-RAGE antibodies of the present invention may be used in combination with one or more additional agents, which may be administered to a subject concurrently or sequentially in either order. The disclosed combination therapies may elicit a synergistic therapeutic effect, Le, an effect greater than the effect of either agent alone. Measurable therapeutic effects are described herein above. For example, a synergistic therapeutic effect may be an effect of at least about two-fold greater than the therapeutic effect elicited by a single agent, or at least about at least about five-fold greater, or at least about ten-fold greater, or at least about twenty-fold greater, or at least about fifty-fold greater, or at least about one hundred-fold greater.
[0176] For example, the invention includes administering a therapeutically effective amount of an antibody that binds specifically to RAGE and inhibits the binding of a RAGE binding partner in combination with another antibody that binds specifically to Aβ. The antibody that binds to Aβ can be an antibody that specifically binds to Aβ peptide without binding to full-length amyloid precursor protein (APP). Alternatively, the antibody of the invention may be administered in combination with antibodies that bind to and/or capture soluble Aβ, or that bind to an amyloid deposit in the patient and induce a clearing response against the amyloid deposit. Such a clearing response can be effected by Fc receptor mediated phagocytosis. Such a clearing response can be engineered into an antibody, for example, by including an Fc receptor-binding domain (e.g., an IgG2a constant region). The antibody of the invention can also be administered to a patient who has received or is receiving an Aβ vaccine. In the case of Alzheimer's and Down's syndrome, in which amyloid deposits occur in the brain, antibodies of the invention can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier. Antibodies of the invention can also be administered in combination with other agents that enhance access of the therapeutic agent to a target cell or tissue, for example, liposomes and the like. Coadministering such agents can decrease the dosage of a therapeutic agent (e.g., therapeutic antibody or antibody chain) needed to achieve a desired effect.
[0177] A variety of RAGE ligands, and particularly those of the S 100/calgranulin family and HMGBl, are produced in inflamed tissues. This observation is true both for acute inflammation, such as that seen in response to a lipopolysaccharide challenge (as in sepsis), and for chronic inflammation. Cardiovascular diseases, particularly those arising from atherosclerotic plaques, are thought to have a substantial inflammatory component and therefore can be treated using the compositions of the invention. Such cardiovascular diseases include, e.g., occlusive, thrombotic and embolic diseases, such as angina, fragile plaque disorder and embolic stroke, respectively, as well as the other cardiovascular diseases described herein
[0178] In another specific embodiment, the present invention provides a method for treating TNF-alpha mediated inflammation and/or a disorder characterized by TNF-alpha mediated inflammation. In still another embodiment, the present invention provides a method for treating a disorder characterized by the presence of DNA immune complexes, including, but not limited to, SLE.The prophylactic or therapeutic agents used in combination with a composition of the invention can also be cyclically administered. Cycling therapy involves the administration of a first prophylactic or therapeutic agent for a period of time, followed by the administration of a second prophylactic or therapeutic agent for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the agents, to avoid or reduce the side effects of one of the agents, and/or to improve the efficacy of the treatment.
[0179] Nonlimiting examples of disorders or conditions which can be usefully treated using the antibody compositions, i.e., pharmaceutical compositions of the present invention, include those disorders or conditions enumerated in the background section of this specification and below. In particular embodiments, the disorder or condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, scleroderma, chronic obstructive pulmonary disease (COPD), rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, disseminated bacteremia, Dengue fever, candidiasis, malaria, fϊlariasis, amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, restenosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, preclinical Alzheimer's disease, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury (SCI), paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget' s disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus (SLE), Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft- versus-host disease, Berger's disease, Type I diabetes, ankylosing spondylitis, Retier's syndrome, or Hodgkin's disease. In more particular embodiments, the disorder or condition is appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, scleroderma, COPD, disseminated bacteremia, burns, preclinical Alzheimer's disease, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, SCI, paralysis, SLE, allograft rejection or graft-versus-host disease.
[0180] As used herein, a "therapeutically effective amount," an "amount sufficient" and like terms refers to that amount of the therapeutic agent, e.g., a RAGE antibody composition of the invention, sufficient to treat or manage a disease or disorder for example, a disorder or condition characterized by TNF-alpha mediated inflammation and/or the presence (or elevated levels) of DNA immune complexes. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of the disease, e.g., delay or minimize the severity of a disease. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of an inflammatory disorder. Further, a therapeutically effective amount with respect to a pharmaceutical composition of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management a disease, e.g., an inflammatory disease. For example, in the case of SLE, a therapeutically effective dose preferably prevents further deterioration of physical symptoms associated with SLE, such as, for example, pain, fatigue or weakness. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. [0181] In one embodiment, the invention is directed to methods of administering and using compositions and antibodies or the invention to treat and/or prevent a disorder or condition selected from the group consisting of appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, hepatitis, Crohn's disease, COPD, scleroderma, asthma, allergy, anaphylactic shock, rheumatoid arthritis, SLE, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, septic abortion, disseminated bacteremia, burns, rheumatoid arthritis, coeliac disease, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, spinal cord injury, paralysis, allograft rejection and graft-versus-host disease, type I diabetes. In certain embodiments, the disorder or condition is endotoxic shock or sepsis. In certain other embodiments, the disorder or condition is SLE. In still other embodiments, the disorder or condition is rheumatoid arthritis.
[0182] Certain embodiments of the invention are directed to methods of administering and using compositions and antibodies of the invention to treat and/or prevent sepsis, lupus and arthritis (e.g., RA, psoriatic arthritis, juvenile rheumatoid arthritis). A specific embodiment of the invention is directed to methods of administering and using compositions and antibodies of the invention to treat and prevent lupus.
[0183] In a specific embodiment, the antibody compositions, e.g., pharmaceutical compositions of the invention, reduce the TNF-alpha mediated inflammation in a mammal by at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90% as compared to a control composition.
[0184] In another specific embodiment, the antibody compositions, i.e., pharmaceutical compositions of the invention reduce a disease index score (e.g., Systemic Lupus Activity Measure (SLAM), Systemic Lupus Erythematosus Disease Activity Index
(SLEDAI)) in a mammal suffering from lupus by at least 5%, or at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90% as compared to a control composition. [0185] As discussed supra, any agent or therapy which is known to be useful, or which has been used or is currently being used for the prevention, management, treatment or amelioration of an inflammatory disorder or one or more symptoms thereof can be used in combination with an antibody composition of the invention. Specific examples of immunomodulatory agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, Cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics {e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes {e.g., leflunamide), anti-T cell receptor antibodies {e.g., anti-CD4 antibodies {e.g., cM-T412 (Boeringer), IDEC-CE9.1.RTM. (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies {e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson)), anti-CD20 antibodies (e.g., Rituxan (IDEC & Genentech, U.S. and International Patent Publications US2004/0202658, WO00/67796) and derivatives there of, HuMax-CD20 (GenMab and
Medarex, U.S. Patent Publication 2004/0167319)), anti-CD19 antibodies {see, e.g., U.S. and international Patent Publications US20020041847, US20030133930 and WO 05/012493), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)), anti-CD2 antibodies (e.g., MEDI-507 (Medlmmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904), anti-CD 1 Ia antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC)); anti-cytokine receptor antibodies (e.g., anti- IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti- TNF-α antibodies, anti-IL-β antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX- IL-8 (Abgenix)), and anti-IL-12 antibodies)); anti-CD22 antibodies (e.g., non- ligand blocking antibodies such as Epratuzumab (Immunomedics) and ligand blocking antibodies (e.g., U.S. Patent Publictions2004/0001828 and 2003/0202975)); CTLA4-immunoglobulin; LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432); soluble cytokine receptors (e.g., the extracellular domain of a TNF-α receptor or a fragment thereof, the extracellular domain of an IL- lβ receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof); cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-Il, IL-12, IL-15, TNF-α, TNF-β, interferon (IFN)-α, IFN-β, IFN-. gamma., and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-α antibodies, and anti-IFN-. gamma. antibodies).
[0186] Non-limiting examples of anti-angiogenic agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include Vitaxin® (Medlmmune) or other anti-alpha v beta3 antibodies (e.g., CNTO95 (Centocor)), endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of platelet factor-4, the anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the small anti-angiogenic peptides of laminin, fibronectin, procollagen and EGF, acid fibroblast growth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF) antagonists, vascular endothelial growth factor (VEGF) antagonists, VEGF receptor (VEGFR) antagonists (e.g., anti- VEGFR antibodies), and Avastin ®. [0187] Non-limiting examples of TNF-α antagonists which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, and antigen-binding fragments thereof) such as antibodies that immunospecifically bind to TNF-α, nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules that blocks, reduces, inhibits or neutralizes the function, activity and/or expression of TNF-α. In various embodiments, a TNF-α antagonist reduces the function, activity and/or expression of TNF-α by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a control such as phosphate buffered saline (PBS). Examples of antibodies that immunospecifically bind to TNF-α include, but are not limited to, infliximab (REMICADE™; Centocor), D2E7 (Abbott
Laboratories/Knoll Pharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known as HUMICADE™ and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), and TN3-19.12 (Williams et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 2762-2766; Thorbecke et al, 1992, Proc. Natl. Acad. Sci. USA 89:7375-7379). The present invention also encompasses the use of antibodies that immunospecifically bind to TNF-α disclosed in the following U.S. patents in the compositions and methods of the invention: U.S. Pat. Nos. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380; 5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272; 5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and 6,171,787. Examples of soluble TNF-α receptors include, but are not limited to, sTNF-Rl (Amgen), etanercept (ENBREL™; Immunex) and its rat homo log RENBREL™, soluble inhibitors of TNF-α derived from
TNFrI, TNFrII (Kohno et al, 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335), and TNF-α Inh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192).
[0188] Other TNF-α antagonists encompassed by the invention include, but are not limited to, IL-10, which is known to block TNF-α production via interferon .gamma.- activated macrophages (Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89:8676-8680),
TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-I (Serono/Yeda), the vaccine CytoTAb (Protherics), antisense molecule 104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC- 333 (Dupont), VX-745 (Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco), NPI- 13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax), auranofm (Ridaura) (SmithKline Beecham Pharmaceuticals), quinacrine (mepacrine dichlorohydrate), tenidap (Enablex), Melanin (Large Scale Biological), and anti-p38 MAPK agents by Uriach.
[0189] Non-limiting examples of anti-inflammatory agents which can be administered in combination with an antibody composition of the invention to a subject with an inflammatory disorder include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta-agonists, anticholingeric agents, and methyl xanthines. Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™), etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™), ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™), sulindac (CLINORIL™), tolmentin (TOLECTIN™.), rofecoxib (VIOXX™), naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON) and nabumetone (RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme (e.g., COX-I and/or COX-2). Examples of steroidal anti- inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes, and leukotrienes. [0190] In specific embodiments, patients with osteoarthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful for osteoarthritis prevention, treatment, management or amelioration including but not limited to: analgesics (non- limiting examples are acetaminophen, in a dose up to 4000 mg/d; phenacetin; and tramadol, in a daily dose in the range of 200 to 300 mg); NSAIDs (non- limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin. Low dose NSAIDs are preferred, e.g., ibuprofen at 1200 mg/d, naproxen at 500 mg/d. A gastroprotective agent, e.g., misoprostol, famotidine or omeprazole, is preferred to use concurrently with a NSAID); nonacetylated salicylates including but not limited to salsalate; cyclooxygenase (Cox)-2-specific inhibitors (CSIs), including but not limited to, celecoxib and rofecoxib; intra- or periarticular injection of a depot glucocorticoid preparation; intra-articular injection of hyaluronic acid; capsaicin cream; copious irrigation of the osteroarthritis knee to flush out fibrin, cartilage shards and other debris; and joint replacement surgery. The antibody compositions of the invention can also be used in combination with other nonpharmacologic measures in prevention, treatment, management and amelioration of osteoarthritis including but not limited to: reduction of joint loading (non- limiting examples are correction of poor posture, support for excessive lumbar lordosis, avoid excessive loading of the involved joint, avoid prolonged standing, kneeling and squatting); application of heat to the affected joint; aerobic exercise and other physical therapies.
[0191] In specific embodiments, patients with rheumatoid arthritis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to: NSAIDs (non-limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.); analgesics (non-limiting examples are acetaminophen, phenacetin and tramadol); CSIs including but not limited to, celecoxib and rofecoxib; glucocorticoids (preferably low-dose oral glucocorticoids, e.g., <7.5 mg/d prednisone, or monthly pulses with high-dose glucocorticoids, or intraarticular glucocorticoids); disease-modifying antirheumatic drugs (DMARDs) including but not limited to, methotrexate (preferably given intermittent low dose, e.g., 7.5-30 mg once weekly), gold compounds (e.g., gold salts), D-penicillamine, the antimalarials (e.g., chloroquine), and sulfasalazine; TNF-α neutralizing agents including but not limited to, etanercept and infliximab; immunosuppressive and cytotoxic agents (examples include but not limited to, azathioprine, leflunomide, cyclosporine, and cyclophosphamide), and surgery (examples include but not limited to, arthroplasties, total joint replacement, reconstructive hand surgery, open or arthroscopic synovectomy, and early tenosynovectomy of the wrist). The antibody compositions of the invention may also be used in combination with other measures in prevention, treatment, management and amelioration of the rheumatoid arthritis including but not limited to: rest, splinting to reduce unwanted motion of inflamed joint, exercise, used of a variety of orthotic and assistive devices, and other physical therapies. The antibody compositions of the invention may also be used in combination with some nontraditional approaches in prevention, treatment, management and amelioration of rheumatoid arthritis including but not limited to, diets (e.g., substituting omega-3 fatty acids such as eicosapentaenoic acid found in certain fish oils for dietary omega-6 essential fatty acids found in meat), vaccines, hormones and topical preparations. [0192] In specific embodiments, patients with chronic obstructive pulmonary disease
(COPD) are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with other agents or therapies useful in prevention, treatment, management and amelioration of COPD including, but not limited to: bronchodilators including but not limited to, short- and long- acting β2-adrenergic agonists (examples of short-acting β2 agonist include but not limited to, albuterol, pirbuterol, terbutaline, and metaproterenol; examples of long-acting β2 agonist include but not limited to, oral sustained-release albuterol and inhaled salmeterol), anticholinergics (examples include but not limited to ipratropium bromide), and theophylline and its derivatives (therapeutic range for theophylline is preferably 10-20 μg/mL); glucocorticoids; exogenous αiAT (e.g., (Xi AT derived from pooled human plasma administered intravenously in a weekly dose of 60 mg/kg ); oxygen; lung transplantation; lung volume reduction surgery; endotracheal intubation, ventilation support; yearly influenza vaccine and pneumococcal vaccination with 23-valent polysaccharide; exercise; and smoking cessation. [0193] In specific embodiments, patients with pulmonary fibrosis are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for pulmonary fibrosis therapy including but not limited to: oxygen; corticosteroids (a non- limiting example is to administer daily prednisone beginning at 1-1.5 mg/kg/d (up to 100 mg/d) for six weeks and tapering slowly over 3-6 months to a minimum maintenance dose of 0.25 mg/kg/d); cytotoxic drugs (non-limiting examples are cyclophosphamide at 100-120 mg orally once daily, and azathioprine at 3 mg/kg up to 200 mg orally once daily); bronchodilators (non-limiting examples are short- and long- acting β2-adrenergic agonists, anticholinergics, and theophylline and its derivatives); and antihistamines (non- limiting examples are diphenhydramine and doxylamine).
[0194] In specific embodiment, patients with SCI are administered prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for SCI therapy including but not limited to: glucocorticoid steroids (a non-limiting example is to administer methylprednisolone 30 mg/kg bolus over 15 minutes and an infusion of methylprednisolone at 5.4 mg/kg/h for 23 hours beginning 45 minutes after the bolus), neuroprotectors (e.g., minocyclin), regeneration therapies (e.g., stem cell treatments, hydrogels), weak electrical fields (e.g., extraspinal oscillating field stimulator implantable medical device). [0195] In specific embodiments, patients with asthma are administered a prophylactically or therapeutically effective amount of an antibody composition of the invention alone or in combination with an effective amount of one or more other agents useful for asthma therapy including but not limited to: adrenergic stimulants (examples include but not limited to, catecholamines, e.g., epinephrine, isoproterenol, and isoetharine; resorcinols, e.g., metaproterenol, terbutaline, and fenoterol; and saligenins, e.g., salbutamol. Inhalation is the preferred route of administration for adrenergic stimulants); methylxanthines including but not limited to theophylline and its various salts; anticholinergics including but not limited to, atropine sulfate, atropine methylnitrate, and ipratropium bromide; glucocorticoids (examples including but not limited to systemic or oral steroids, and inhaled glucocorticoids); mast cell stabilizing agents (examples include but not limited to, cromolyn sodium and nedocromil sodium); leukotriene modifiers (examples include but not limited to, Zileuton, zafirlukast and montelukast); immunosuppressant agents (examples include but not limited to, methotrexate and gold salts); and mucolytic agents (examples include but not limited to acetylcysteine). [0196] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
[0197] In one embodiment, the pharmaceutical compositions of the invention are pyrogen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, even trace amounts of harmful and dangerous endotoxin must be removed. In certain specific embodiments, the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. [0198] When used for in vivo administration, the compositions described herein should be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes or by other means well known in the art. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Company, Easton, PA, 1990). Compositions comprising antibodies, such as those disclosed herein, ordinarily will be stored in lyophilized form or in solution. It is contemplated that sterile compositions comprising antibodies of the invention are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle. [0199] In one embodiment, the composition of the present invention may be administered orally, parenterally, i.e., including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques, by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
[0200] In another embodiment, in accordance with the method of the present invention, said composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
[0201] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below. [0202] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[0203] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb. [0204] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et ah, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid) [0205] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et ah, 1980, Surgery 88:507; Saudek et ah, 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sd. Rev. Macromol. Chem. 23:61; see also Levy et ah, 1985, Science 228:190; During et ah, 1989, Ann. Neurol. 25:351; Howard et ah, 1989, J. Neurosurg. 71 :105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).
[0206] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment {e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et ah, 1991, Proc. Natl. Acad. Sci. USA 88:1864- 1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination. [0207] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[0208] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [0209] The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. [0210] The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[0211] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half- life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, Hpidation.
[0212] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. [0213] The excipient included with the polypeptide in these compositions is chosen based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the disorder or condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as endotoxic shock, and oral administration may be preferred to treat a gastrointestinal disorder such as a gastric ulcer. The route of administration and the dosage of the composition to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the disorder/condition or disorders/conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. Thus, depending on the disorder or condition, the antibody composition can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabuccaly and transdermally to the patient. [0214] Accordingly, compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
[0215] Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
[0216] The compositions of the present invention can easily be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the antibody compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as, for example, benzyl alcohol or methyl parabens, antioxidants such as, for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. [0217] Rectal administration includes administering the pharmaceutical compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120C, dissolving the antibody composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
[0218] Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves and the like.
[0219] The antibody compositions described herein can also include an antagonist of an early sepsis mediator. As used herein, an early sepsis mediator is a proinflammatory cytokine that is released from cells soon (i.e., within 30-60 min.) after induction of an inflammatory cytokine cascade (e.g., exposure to LPS). Nonlimiting examples of these cytokines are TNF, IL-lα, IL-lβ, IL-6, PAF, and MIF. Also included as early sepsis mediators are receptors for these cytokines (for example, tumor necrosis factor receptor type 1) and enzymes required for production of these cytokines, for example, interleukin-lβ converting enzyme). Antagonists of any early sepsis mediator, now known or later discovered, can be useful for these embodiments by further inhibiting an inflammatory cytokine cascade.
[0220] Nonlimiting examples of antagonists of early sepsis mediators are antisense compounds that bind to the mRNA of the early sepsis mediator, preventing its expression (see, e.g., Ojwang et al., 1997, Biochemistry 36:6033-6045; Pampfer et al., 1995, Biol. Reprod. 52:1316-1326; U.S. Patent No. 6,228,642; Yahata et al, 1996, Antisense Nucleic Acid Drug Dev. 6:55-61; and Taylor et al., 1998, Antisense Nucleic Acid Drug Dev. 8:199-
205), ribozynies that specifically cleave the mRNA of the early sepsis mediator (see, e.g., Leavitt et al., 2000, Antisense Nucleic Acid Drug Dev. 10: 409-414; Kisich et al., 1999; and Hendrix et al., 1996, Biochem. J. 314: 655-661), and antibodies that bind to the early sepsis mediator and inhibit their action (see, e.g., Kam and Targan, 2000, Expert Opin. Pharmacother. 1 : 615-622; Nagahira et al., 1999, J. Immunol. Methods 222, 83-92; Lavine et al., 1998, J. Cereb. Blood Flow Metab. 18: 52-58; and Holmes et al., 2000, Hybridoma 19: 363-367). Any antagonist of an early sepsis mediator, now known or later discovered, is envisioned as within the scope of the invention. The skilled artisan can determine the amount of early sepsis mediator to use in these compositions for inhibiting any particular inflammatory cytokine cascade without undue experimentation with routine dose-response studies.
4.6 Diagnostics and Imaging
[0221] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression. [0222] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
[0223] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art {e.g., see Jalkanen, et al., 1985, J. Cell. Biol. 101 :976-985; Jalkanen, et al, 1987, J. Cell. Biol.
105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). [0224] Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003). [0225] One embodiment of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: (a) administering (for example, parenterally, subcutaneous Iy, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; (b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); (c) determining background level; and (d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
[0226] Also as described herein, antibodies of the invention may be used to treat, diagnose, or prognose an individual having sepsis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., RA, psoriatic arthritis, juvenile rheumatoid arthritis), anaphylactic shock, organ ischemia, reperfusion injury, and allograft rejection, systemic lupus erythematosus, Type I diabetes and related pathological states, including diabetic vasculopathy, nephropathy, retinopathy, neuropathy, Alzheimer's disease and immune/inflammatory reactions of blood vessel walls and other RAGE -mediated conditions or disorders.
[0227] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et ah, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.
Rhodes, eds., Masson Publishing Inc. (1982).
[0228] Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours.
In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
[0229] In one embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
[0230] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[0231] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et ah, U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
5. SPECIFIC EMBODIMENTS
[0232] 1. An isolated monoclonal antibody that specifically binds the human RAGE polypeptide of SEQ ID NO:1, wherein said antibody inhibits the binding of an HMGBl-CpG complex to RAGE. [0233] 2. The antibody of embodiment 1, wherein said antibody binds amino acid residues 24-129 of SEQ ID NO:1.
[0234] 3. The antibody of embodiment 1 , wherein said antibody binds amino acid residues 31 - 106 of SEQ ID NO : 1. [0235] 4. The antibody of embodiment 1 , wherein said antibody binds amino acid residues 1-121 of SEQ ID NO:l.
[0236] 5. The antibody of embodiment 1, wherein said antibody binds amino acid residues 235-336 of SEQ ID NO: 1. [0237] 6. The antibody of embodiment 1, wherein said antibody binds amino acid residues 234-342 of SEQ ID NO:1.
[0238] 7. The antibody of embodiment 1 , wherein said antibody binds amino acid residues 252-308 of SEQ ID NO:1.
[0239] 8. An isolated monoclonal antibody that specifically binds human RAGE, comprising:
[0240] a) a light chain variable region having the 3 CDRs of the light chain of the antibody S2, SEQ ID NO:4, and a heavy chain variable region; or
[0241] b) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody S2, SEQ ID NO:5, and a light chain variable region; or [0242] c) a light chain variable region having the 3 CDRs of the light chain of the antibody L30, SEQ ID NO:6, and a heavy chain variable region; or
[0243] d) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L30, SEQ ID NO:7, and a light chain variable region; or
[0244] e) a light chain variable region having the 3 CDRs of the light chain of the antibody L37, SEQ ID NO:8, and a heavy chain variable region; or
[0245] f) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L37, SEQ ID NO: 9, and a light chain variable region;
[0246] wherein said antibody inhibits the binding of an HMGB 1 -CpG complex to RAGE. [0247] 9. The antibody of embodiment 8, wherein said antibody comprises a heavy chain variable region selected from the group consisting of the antibody S2, SEQ ID NO:5, antibody L30, SEQ ID NO:7, and the antibody L37, SEQ ID NO:9.
[0248] 10. The antibody of embodiment 8, wherein said antibody comprises a light chain variable region selected from the group consisting of the antibody S2, SEQ ID NO:4, antibody L30, SEQ ID NO:6, and the antibody L37, SEQ ID NO:8.
[0249] 11. The antibody of embodiment 8, wherein said antibody comprises
[0250] a) a light chain variable region from the antibody S2, SEQ ID NO:4 and a heavy chain variable region from the antibody S2, SEQ ID NO:5, or [0251] b) a light chain variable region from the antibody L30, SEQ ID NO:6 and a heavy chain variable region from the antibody L30, SEQ ID NO:7, or
[0252] c) a light chain variable region from the antibody L37, SEQ ID NO: 8 and a heavy chain variable region from the antibody L37, SEQ ID NO:9. [0253] 12. The antibody of any of the preceding embodiments, wherein said antibody selectively inhibits the binding of one or more of RAGE ligands selected from the group consisting of AGEs, S100A12, SlOOAl, S100A4, SlOOAI l, S100A13, SlOOB, SlOOP, amyloid- β-pep tide and Mac-1.
[0254] 13. The antibody of any of the preceding embodiments, wherein said antibody does not inhibit the binding of the human S100A12 polypeptide.
[0255] 14. The antibody of any of the preceding embodiments, wherein said antibody does not inhibit the binding of the bovine SlOOB polypeptide.
[0256] 15. The antibody of any of the preceding embodiments, wherein said antibody inhibits mammalian cell RAGE-mediated expression of one or more Type I Interfereon (IFN) genes or one or more type-I IFN-inducible genes selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35.
[0257] 16. The antibody of embodiment 15, wherein said antibody inhibits said expression by at least 25%.
[0258] 17. The antibody of any of the preceding embodiments, wherein said antibody inhibits the intracellular or extracellular localization of a RAGE molecule with a TLR.
[0259] 18. The antibody of embodiment 17, wherein said TLR is selected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRIl. [0260] 19. The antibody of any of the preceding embodiments, wherein the antibody is selected from the group consisting of: a human antibody, a humanized antibody; a chimeric antibody; a single-chain Fv (scFv); an Fab fragment; an F(ab') fragment; an intrabody; and a synthetic antibody.
[0261] 20. An antibody selected from the group consisting of: S2/1.50#2 (S15), S2/2.2#l (S22), S2/4.146#18 (S41), S2/4.17#4 (S2), SNSR2.62#5 (N262), LpxR4.12#2 (L4), LpxR30#12 (L30) and LpxR37#2 (L37).
[0262] 21. An antibody that binds the same epitope as the antibody of any one of embodiments 1-20. [0263] 22. An antibody that competes with an antibody of any one of embodiments 1-20 for binding to the human RAGE polypeptide of SEQ ID NO: 1.
[0264] 23. An antibody comprising at least one CDR derived from an antibody of embodiment 20, wherein said antibody binds the human RAGE polypeptide of SEQ ID NO: 1 and inhibits the binding of an HMGBl-CpG complex to RAGE.
[0265] 24. The antibody of embodiment 8 or 23, wherein said antibody further comprises a human framework region.
[0266] 25. A composition comprising an antibody of any one of embodiments 1 to 24, and a pharmaceutically acceptable excipient. [0267] 26. A method of reducing TNF-alp ha mediated inflammation in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0268] 27. A method of treating rheumatoid arthritis in a subject comprising administering an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0269] 28. The method of embodiment 27, further comprising administering one or more additional agents useful for the treatment of rheumatoid arthritis, to thereby elicit a synergistic effect.
[0270] 29. A method for treating a condition characterized by the increased presence of DNA immune complexes in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0271] 30. A method for treating systemic lupus erythematosus in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0272] 31. The method of embodiment 30, further comprising administering one or more additional agents useful for the treatment of systemic lupus erythematosus, to thereby elicit a synergistic effect.
[0273] 32. A method for reducing RAGE-mediated expression of one or more type-I IFN-inducible gene selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35 in a subject, comprising administering to the subject an effective amount of a composition comprising the antibody of any one of embodiments 1 to 24. [0274] 33. A method for treating an HMGB 1 -mediated condition in a subj ect, comprising administering an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0275] 34. The method of embodiment 33, further comprising administering one or more additional agents useful for the treatment of an HMGBl -mediated condition, to thereby elicit a synergistic therapeutic effect.
[0276] 35. The method of embodiment 33 or 34, wherein the HMGB 1 -mediated condition is selected from the group consisting of, sepsis, systemic lupus erythematosus, inflammatory lupus nephritis, scleroderma, COPD, psoriasis, Sjogren's disease, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, type I diabetes, allograft rejection and graft-versus-host disease.
[0277] 36. A method for treating a disease or disorder characterized by amyloid deposit of A-beta in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0278] 37. The method of embodiment 36, wherein the disease or disorder is characterized by amyloid deposit of A-beta in brain.
[0279] 38. The method of embodiment 36, wherein the disease or disorder is Alzheimer's disease. [0280] 39. The method of embodiment 36, wherein the disease or disorder is preclinical Alzheimer's disease.
[0281] 40. The method of any one of embodiments 36-39, further comprising administering one or more additional agents useful for the treatment of Alzheimer's disease, to thereby elicit a synergistic therapeutic effect. [0282] 41. A method of inhibiting or reducing accumulation of amyloid deposit of
A-beta in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of embodiments 1 to 24.
[0283] 42. The method of embodiment 41 , comprising inhibiting or reducing accumulation of amyloid deposit of A-beta in brain. [0284] 43. The method of embodiment 42, wherein the accumulation of amyloid deposit of A-beta in brain is associated with Alzheimer's disease.
[0285] 44. The method of embodiment 42, wherein the accumulation of amyloid deposit of A-beta in brain is associated with preclinical Alzheimer's disease. [0286] 45. A method of inhibiting or reducing neurodegeneration in a subj ect, comprising administering to the subject an effective amount of the antibody of any one of embodiments 1 to 24.
[0287] 46. The method of embodiment 45, comprising inhibiting or reducing neurodegeneration in brain.
[0288] 47. The method of embodiment 45 , wherein the neurodegeneration is associated with Alzheimer's disease.
[0289] 48. The method of embodiment 45, wherein the neurodegeneration is associated with preclinical Alzheimer's disease. [0290] 49. The method of any one of embodiments 45-48, further comprising administering one or more additional agents useful for inhibiting or reducing neurodegeneration, to thereby elicit a synergistic effect.
[0291] 50. A method of inhibiting or reducing cognitive decline, or improving cognition, in a subject, comprising administering to the subject an effective amount of the antibody of any one of embodiments 1 to 24.
[0292] 51. The method of embodiment 50, wherein the cognitive decline is associated with Alzheimer's disease.
[0293] 52. The method of embodiment 50, wherein the cognitive decline is associated with preclinical Alzheimer's disease. [0294] 53. The method of embodiment 50, 51 or 52, further comprising administering one or more additional agents useful for inhibiting or reducing cognitive decline, or improving cognition, to thereby elicit a synergistic effect.
[0295] 54. The method of any one of embodiments 26 - 53, wherein the subject is a human subject.
6. EXAMPLES
[0296] The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
6.1 Example 1.
Generation of anti-RAGE Antibodies Materials and Methods
[0297] Antibody Generation: The extracellular domain of human RAGE (huRAGE) fused to Fc region of human IgGl was expressed in NSO cells and used to immunize BALB/c mice. Three groups of mice were immunized as follows: Group I - footpad immunization using TiterMax adjuvant; Group II - i.p. immunization using CFA/IFA adjuvant; Group III - i.p. immunization using TiterMax adjuvant. Hybridomas were generated using standard methodologies. Clones were initially screened for IgG reactivity specific for the huRAGE-Fc fusion protein used for immunization in an ELISA format followed by FACS studies using cells stably (NSO) or transiently (293F) transfected with full-length huRAGE. Positive clones expressing anti-RAGE mAbs were then further purified by limited dilution cloning. The hybridomas are grown in DMEM/2% ultra low IgG serum and the mAbs are purified by protein G chromatography.
[0298] Bindins Studies and Epitope Mapping: The cross-reactivity of anti-huRAGE mAbs against murine RAGE (mRAGE) was examined by ELISA. Epitope mapping of anti- RAGE mAbs was performed using 293F cells expressing the different human RAGE domain deletion mutants shown in Figure 2: V domain (amino acids 1-121), Cl domain (amino acids 122-233), C2 domain (amino acids 234-342), VCl domains (amino acids 1-233), and C1C2 domains (amino acids 122-342) of GenBank accession number BC020669. For surface expression of these constructs, the signal sequence (amino acids 1-26) was added to the N- terminal of Cl, C2, and C1C2 constructs, and the transmembrane-cytoplasmic tail sequence (amino acids 343-404) was included or added to the C-terminal of all constructs. Briefly, 293F cells expressing huRAGE and its mutants were incubated with anti-huRAGE mAbs, followed by PE-conjugated goat anti-mouse IgG Fc-specifϊc Fab fragments and analyzed by FACS. Representative data are shown in Figure 3 and summarized in Table 2. [0299] Inhibition of RAGE-HMGB 1/CpG complexes: The ability of anti-huRAGE mAbs to inhibit the binding of HMGB- 1/CpG to immobilized huRAGE was examined using in vitro assays. Briefly, 96-well plates were coated with 50 μL of 16 nM huRAGE -Fc (or 1.0 mg/ml huRAGE-Fc), the plates were then blocked and incubated with serially diluted anti- RAGE mAbs for 1 hr. HMGBl/CpG complex at 1 : 1 molar ratio was then added to the wells at a final concentration of 30 nM (or 0.8 mg/ml) and incubated at 370C for 1 hr. The wells were then washed, and incubated with 3 nM (or 0.5mg/ml) of biotinylated anti-HMGBl antibody (S16-biotin), followed by detection with streptavidin-HRP. Representative data are shown in Figure 4 and summarized in Table 2 and Table 5. [0300] Surface Plasmon Resonance Measurements: All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden). The ligand, human RAGE/Fc, was prepared at 5 ug/mL in 1OmM NaOAc, pH4 buffer, then injected onto an EDC/NHS-activated CM5 sensor chip (BIAcore, Inc. Uppsala, Sweden) using a standard immobilization protocol. Following this, unreacted active ester moieties were quenched by injecting IM Et-NH2 (ethanolamine) over the sensor chip surface. These coupling reagents were also purchased from the manufacturer (BIAcore, Inc.). A total of 573 resonance units (RUs) of huRAGE/Fc remained bound to the sensor chip surface after the coupling procedure. Separately, a blank surface was also prepared on the sensor chip using the identical protocol, minus the protein. Several buffer injections were also interspersed throughout the injection series. Later, these buffer injections were used, along with the reference cell data, to correct the raw data sets for injection artifacts and/or non-specific 'binding' through a technique commonly referred to as "double-referencing." (D. G. Myszka, Improving biosensor analysis. J. MoI. Recognit. 12 (1999), pp. 279-284). [0301] For the kinetic experiments, IgGs were prepared as two or three fold dilution series, as follows: L30 (0.0391 nM - 40 nM) in HBS-EP buffer (BIAcore, Inc., consisting of the following: 1OmM HEPES buffer, pH7.4, 15OmM NaCl, 3mM EDTA, and 0.005% P20). A duplicate injection of each concentration of the IgGs was then injected over both the huRAGE/Fc and reference cell surfaces, which were connected in series. Between injections, the bound IgG was removed from the huRAGE-Fc surface with two, one-minute injections of 1O mM GIy buffer, pHl.5.
[0302] Raw binding data was corrected in the manner described by Myszka (D. G. Myszka, Improving biosensor analysis. J. MoI. Recognit. 12 (1999), pp. 279-284). Fully corrected binding data was then globally fit using a 1 : 1 binding model (BIAevaluation 4.1 software, BIAcore, Inc, Uppsala, Sweden) to obtain the kinetic rate and apparent binding constants. The Kd value determined for the L30 mAb is shown in Table 6.
[0303] Determination ofEC50 values: The anti-RAGE antibodies were tested to determine EC50 values for huRAGE-His or huRAGE-Fc binding by ELISA, in accordance with a protocol similar to the protocol shown below. The results are shown in Table 6. Protocol: a. Coat 96-well plate with 100 μl of 1 μg/ml of huRAGE-His or huRAGE-Fc in PBS over night at 40C. b. Block huRAGE-His or huRAGE-Fc coated plate with 200 μl PBS/4% skim milk for 1 hr at room temperature. c. Wash 4 times with PBS/0.1 % Tween-20. d. Add 50 μl of mouse anti-RAGE mAb (starting at 20 μg/ml and then serially diluted 4- fold) to huRAGE-His coated well. Incubate at room temperature for 1 hr. e. Wash 4 times with PBS/0.1 % Tween-20. f. Add 50 μl of rabbit anti-mouse IgG Fc-HRP (minimum cross reactivity to human and bovine serum proteins). Incubate for 1 hr at room temperature. g. Wash 4 times with PBS/0.1% Tween-20. h. Add 50 μl of TMB to develop the plate for 10 min at RT. i. Add 50 μl to stop the reaction. j. Read the plate at 450 nm. k. The EC50 value of each mAb is obtained by plotting OD (450 nm) vs. mAb concentration using the GraphPad Prism program (GraphPad Software, San Diego, CA).
Results
[0304] A huRAGE-Fc fusion construct was used to generate anti-huRAGE antibodies. 21 anti-huRAGE mAbs were identified. Several were found to have low expression levels and were not further characterized. The anti-huRAGE reactivity of 16 antibodies was characterized by ELISA and FACS analysis. At least one anti-huRAGE antibody cross-reacts with mRAGE by ELISA. The reactive domains of the anti-huRAGE mAbs have been determined by FACS analysis of anti-huRAGE mAbs to 293F cells expressing different huRAGE deletion mutants. While initial epitope mapping indicated that L37 is a V domain binder, subsequent experiments suggest that it may actually be interacting with the C1/C2 domain of RAGE. Several anti-huRAGE mAbs were shown to inhibit
HMGB- 1/CpG binding to immobilized huRAGE in in vitro inhibition assays. The binding and activity characteristics of the tested antibodies are summarized in Table 2. Following the generation of the data presented in Tables 2 and 5, sequence analyses of the immunoglobulin genes of the L4 and L30 clones revealed that they are separate isolates of the same clone. Subsequent experiments were carried out on antibody from the L30 isolate. The nucleotide and amino acid sequences for the light and heavy chain variable regions of three different clones, S2, L30 and L37, are provided in Figure 7. 6.2 Example 2.
Use of anti-RAGE Antibodies
Materials and Methods
[0305] IFNa gene signature: PBMCs from healthy donors were stimulated for 4h with 50% sera from SLE patients. Total RNA was purified and expression of type I IFN- inducible genes, including DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, IFI35 were measured by real-time QRT-PCR analysis.
[0306] Inhibition of IFNa-induced gene expression: The antibodies were also tested for their ability to inhibit the IFNα gene signature profile. As follows, PBMCs from healthy donors were stimulated for 4h with 50% sera from SLE patients in the presence of anti- huRAGE antibodies or an unrelated control antibody at a concentration of 10 μg/mL. In addition a RAGE-Fc fusion molecule was used as a positive control. The inhibition values are normalized to the negative control Ab. Following incubation the expression profile was tested as described above. The representative data are shown in Figures 5A-B and the averaged results from three repeats are summarized in Table 2. Specific results shown in Figure 6 demonstrate that the L30 (LpxR30#12) antibody inhibits the IFN-α gene signature induced by anti-dsDNA+ SLE serum.
[0307] Inhibition of immune complex induced IFNa secretion in PBMC: Cell lysate of mouse embryonic fibroblast (MEF) was prepared by subjecting cells at lxlO7/ml to 4 cycles of freeze/thaw. The cell lysate was then cleared of insoluble debris by centrifugation and filtered through a 5 -micron filter. Immune complex was formed by mixing 5% of MEF cell lysate with 1% of SLE serum in culture medium. 200 μl of immune complex solution was then added to 4x10Λ5 PBMCs from healthy donors and incubated for 24 hours, in the presence or absence of anti-RAGE antibody. The level of IFN-α in the culture supernatant was measured by ELISA. The results are summarized in Table 2 and Table 5.
Results
[0308] SLE serum can stimulate IFNα gene signature (up-regulation of IFN inducible genes) in PBMCs. This system can be used to examine the ability of therapeutic agents to modulate the IFNα gene signature, i.e., reduce the up-regulation of IFN inducible genes. This system was used to examine the anti-huRAGE antibodies described above (see Example 1). The data summarized in Table 2 and detailed in Table 5 indicate that several anti-RAGE mAbs inhibited the IFNα gene signature by - 45-50 % which was comparable to the inhibition by soluble RAGE-Fc (54±11%). Interestingly, these mAbs also inhibit the binding between RAGE and HMGBl complexed with CpG DNA. Furthermore, these mAbs all bind to RAGE at the C2 domain (plasma-membrane proximal Ig-loop domain). Recent studies on the interaction between SlOOAl 2 (also known as Calgranulin C) and RAGE by Xie et al. (2007, J. Biol. Chem., 282:4218-31), summarized in Table 3, suggest that S100A12 likely binds to the Cl domain of RAGE. Thus, while the RAGE C2 domain antibodies inhibit HMGB 1/CpG complex binding they may not inhibit the binding of SlOOAl 2. In addition, one antibody, LpxR37, was shown to block IC (SLE sera + MEF) induced IFNα in PBMC.
[0309] These results suggest that RAGE and HMGBl contribute to the activation of the mononuclear cells in SLE, and that antibodies targeting RAGE may present a novel therapeutic approach in the treatment of SLE.
Figure imgf000085_0001
a: RAGE domains expressed on 293 cells. b: 1% SLE serum + 5% MEF lysate stimulate huPBMC, measure IFNa secretion. c: 50% SLE serum stimulate huPBMC, measure type I IFN gene signature n=3 expt. d: ELISA using plate-bound RAGE-Fc and HMGB 1/CpG complex, detection by anti-HMGBl (S16). e: cell surface staining of huPBMC pre-incubated with 50% SLE serum for 4 hours. f: sequence analysis showed that these two clones (L4 and L30) were identical.
Table 3. Summary of S100A12 binding to RAGE domains
Figure imgf000086_0001
Table 4. Legend for Sequence Listing
SEQ SEQ
ID DESCRIPTION ID DESCRIPTION
NO: NO:
1 Human RAGE (Ace. No. BC020669) 9 VH amino acid sequence of L37
2 Rat RAGE (Ace. No. NP_445788) 10 VL nucleotide sequence of S2
3 Bovine RAGE (Ace. No. NMJ 73982) 11 VH nucleotide sequence of S2 4 VL amino acid sequence of S2 12 VL nucleotide sequence of L30 5 VH amino acid sequence of S2 13 VH nucleotide sequence of L30 6 VL amino acid sequence of L30 14 VL nucleotide sequence of L37
7 VH amino acid sequence of L30 15 VH nucleotide sequence of L37 VL amino acid sequence of L37
Table 5. Summary of Anti-RAGE Ab properties.
Figure imgf000086_0002
Figure imgf000087_0001
aEC50 values determined against huRAGE-His by ELISA, except as noted in . bEC50 value determined against huRAGE-Fc by ELISA cValues determined against huRAGE-Fc by surface plasmon resonance.
[0310] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes. In addition, the following United States provisional patent applications: 60/984,054 entitled "Anti-RAGE Antibodies and Methods of Use Thereof filed October 31, 2007; 60/946,784 entitled "Anti-RAGE Antibodies and Methods of Use Thereof filed June 28, 2007; 60/9155,578 entitled "Anti-RAGE Antibodies and Methods of Use Thereof filed May 2, 2007; and 60/822,041 entitled "Antagonists of HMGBl and/or RAGE and Methods of Use Thereof filed August 10, 2006; and PCT patent application PCT/US2006/061257 entitled "Antagonists of HMGBl and/or RAGE and Methods of Use Thereof filed November 27, 2006 are incorporated by reference in their entirety.

Claims

1. An isolated monoclonal antibody that specifically binds amino acid residues 24-129, or 31-106, or 1-121, or 235-336, or 234-342, or 252-308, of the human RAGE polypeptide of SEQ ID NO:1, wherein said antibody inhibits the binding of an HMGBl-CpG complex to RAGE.
2. An isolated monoclonal antibody that specifically binds human RAGE, comprising: a) a light chain variable region having the 3 CDRs of the light chain of the antibody S2, SEQ ID NO:4, and a heavy chain variable region; or b) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody S2, SEQ ID NO:5, and a light chain variable region; or c) a light chain variable region having the 3 CDRs of the light chain of the antibody S2, SEQ ID NO:4, and a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody S2, SEQ ID NO:5; or d) a light chain variable region having the 3 CDRs of the light chain of the antibody L30, SEQ ID NO:6, and a heavy chain variable region; or e) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L30, SEQ ID NO:7, and a light chain variable region; or f) a light chain variable region having the 3 CDRs of the light chain of the antibody L30, SEQ ID NO:6, and a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L30, SEQ ID NO:7; or g) a light chain variable region having the 3 CDRs of the light chain of the antibody L37, SEQ ID NO:8, and a heavy chain variable region; or h) a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L37, SEQ ID NO:9, and a light chain variable region; or i) a light chain variable region having the 3 CDRs of the light chain of the antibody L37, SEQ ID NO:8, and a heavy chain variable region having the 3 CDRs of the heavy chain of the antibody L37, SEQ ID NO:9; wherein said antibody inhibits the binding of an HMGBl-CpG complex to RAGE.
3. The antibody of claim 2, wherein said antibody further comprises a human framework region.
4. The antibody of any of the preceding claims, wherein said antibody selectively inhibits the binding of one or more of RAGE ligands selected from the group consisting of AGEs, S100A12, SlOOAl, S100A4, SlOOAI l, S100A13, SlOOB, SlOOP, amyloid-β-peptide and Mac-1.
5. The antibody of any of the preceding claims, wherein said antibody inhibits mammalian cell RAGE-mediated expression by at least 25% of one or more type-I IFN-inducible gene selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35.
6. The antibody of any of the preceding claims, wherein said antibody inhibits the intracellular or extracellular localization of a RAGE molecule with at least one TLRselected from the group consisting of TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRl 1.
7. The antibody of any of the preceding claims, wherein the antibody is selected from the group consisting of: a human antibody, a humanized antibody; a chimeric antibody; a single-chain Fv (scFv); an Fab fragment; an F(ab') fragment; an intrabody; and a synthetic antibody.
8. An antibody selected from the group consisting of: S2/1.50#2 (S15), S2/2.2#l (S22), S2/4.146#18 (S41), S2/4.17#4 (S2), SNSR2.62#5 (N262), LpxR4.12#2 (L4), LpxR30#12 (L30) and LpxR37#2 (L37).
9. A composition comprising an antibody of any one of claims 1 to 8, and a pharmaceutically acceptable excipient.
10. A method of treating a disorder in a subject, wherein said disorder is a) rheumatoid arthritis; or b) systemic lupus erythematosus; or c) characterized by the increased presence of DNA immune complexes; or d) an HMGB 1 -mediated condition; or e) characterized by amyloid deposit of A-beta; said method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of claims 1 to 8.
11. The method of claim 10, wherein said disorder is characterized by amyloid deposit of A- beta, selected from the group consisting of amyloid deposit of A-beta in brain, preclinical Alzheimer's disease, and Alzheimer's disease.
12. The method of claim 10, wherein the disorder is an the HMGBl -mediated condition is selected from the group consisting of, sepsis, systemic lupus erythematosus, inflammatory lupus nephritis, scleroderma, COPD, psoriasis, Sjogren's disease, sepsis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, type I diabetes, allograft rejection and graft-versus-host disease.
13. The method of any one of claims 10 to 12, further comprising administering one or more additional agents useful for the treatment of the disorder, to thereby elicit a synergistic effect.
14. A method of reducing TNF-alpha mediated inflammation in a subject comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of claims 1 to 8.
15. A method for reducing RAGE-mediated expression of one or more type-I IFN-inducible gene selected from the group consisting of: DDX59, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35 in a subject, comprising administering to the subject an effective amount of a composition comprising the antibody of any one of claims 1 to 8.
16. A method of treating a subject comprising a) inhibiting or reducing accumulation of amyloid deposit of A-beta; or b) inhibiting or reducing neurodegeneration; or c) inhibiting or reducing cognitive decline; or d) improving cognition; comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of any one of claims 1 to 8.
17. The method of claim 16, further comprising administering one or more additional agents useful for treating said subject.
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2116556A1 (en) 2008-05-09 2009-11-11 Abbott GmbH & Co. KG Antibodies to receptor of advanced glycation end products (rage) and uses thereof
US20100172896A1 (en) * 2001-03-16 2010-07-08 Marco E Bianchi Hmgb1 protein inhibitors and/or antagonists for the treatment of vascular diseases
EP2308896A1 (en) * 2009-10-09 2011-04-13 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
WO2011042548A1 (en) * 2009-10-09 2011-04-14 Sanofi-Aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
EP2319871A1 (en) * 2009-11-05 2011-05-11 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
US8398977B2 (en) 2007-06-14 2013-03-19 Galactica Pharmaceuticals, Inc. Rage fusion proteins
US8916152B2 (en) 2010-06-14 2014-12-23 Lykera Biomed Sa S100A4 antibodies and therapeutic uses thereof
WO2015085097A1 (en) 2013-12-05 2015-06-11 The Broad Institute, Inc. Compositions and methods for identifying and treating cachexia or pre-cachexia
WO2016201368A1 (en) 2015-06-10 2016-12-15 The Broad Institute Inc. Antibodies, compounds and screens for identifying and treating cachexia or pre-cachexia
WO2017106196A1 (en) * 2015-12-14 2017-06-22 The Broad Institute, Inc. Compositions and methods for treating cardiac dysfunction
EP3733701A1 (en) 2015-03-31 2020-11-04 MedImmune Limited A novel il33 form, mutated forms of il33, antibodies, assays and methods of using the same
WO2021089559A1 (en) 2019-11-04 2021-05-14 Medimmune Limited Anti il-33 therapeutic agent fpr treating renal disorders
WO2021089563A1 (en) 2019-11-04 2021-05-14 Medimmune Limited Methods of using il-33 antagonists

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050026811A1 (en) * 2003-05-20 2005-02-03 Mjalli Adnan M. M. Rage antagonists as agents to reverse amyloidosis and diseases associated therewith
US20060140933A1 (en) * 2002-08-16 2006-06-29 Wyeth And Imperial College Innovations Limited Compositions and methods for treating rage-associated disorders

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060140933A1 (en) * 2002-08-16 2006-06-29 Wyeth And Imperial College Innovations Limited Compositions and methods for treating rage-associated disorders
US20050026811A1 (en) * 2003-05-20 2005-02-03 Mjalli Adnan M. M. Rage antagonists as agents to reverse amyloidosis and diseases associated therewith

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100172896A1 (en) * 2001-03-16 2010-07-08 Marco E Bianchi Hmgb1 protein inhibitors and/or antagonists for the treatment of vascular diseases
US8398977B2 (en) 2007-06-14 2013-03-19 Galactica Pharmaceuticals, Inc. Rage fusion proteins
US9399668B2 (en) 2007-06-14 2016-07-26 Galactica Pharmaceuticals, Inc. Nucleic acids encoding rage fusion proteins
US9066927B2 (en) 2007-06-14 2015-06-30 Galactica Pharmaceuticals, Inc. Methods of treatment using rage fusion proteins
WO2009136382A2 (en) * 2008-05-09 2009-11-12 Abbott Gmbh & Co. Kg Antibodies to receptor of advanced glycation end products (rage) and uses thereof
WO2009136382A3 (en) * 2008-05-09 2010-03-25 Abbott Gmbh & Co. Kg Antibodies to receptor of advanced glycation end products (rage) and uses thereof
EP3059248A1 (en) 2008-05-09 2016-08-24 Abbvie Deutschland GmbH & Co. KG Antibodies to receptor of advanced glycation end products (rage) and uses thereof
US9394363B2 (en) 2008-05-09 2016-07-19 AbbVie Deutschland GmbH & Co. KG Antibodies to receptor of advanced glycation end products (RAGE) and uses thereof
JP2011522519A (en) * 2008-05-09 2011-08-04 アボット ゲーエムベーハー ウント カンパニー カーゲー Antibody against terminal glycation end product receptor (RAGE) and use thereof
EP2500361A1 (en) 2008-05-09 2012-09-19 Abbott GmbH & Co. KG Antibodies to receptor of advanced glycation end products (rage) and uses thereof
US8323651B2 (en) 2008-05-09 2012-12-04 Abbott Laboratories Antibodies to receptor of advanced glycation end products (RAGE) and uses thereof
EP2116556A1 (en) 2008-05-09 2009-11-11 Abbott GmbH & Co. KG Antibodies to receptor of advanced glycation end products (rage) and uses thereof
EP2308896A1 (en) * 2009-10-09 2011-04-13 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
JP2013507115A (en) * 2009-10-09 2013-03-04 サノフイ Polypeptides for binding to "terminal glycation end product receptors" and compositions and methods comprising them
CN102686611A (en) * 2009-10-09 2012-09-19 赛诺菲 Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
WO2011042548A1 (en) * 2009-10-09 2011-04-14 Sanofi-Aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
EP2319871A1 (en) * 2009-11-05 2011-05-11 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
US8916152B2 (en) 2010-06-14 2014-12-23 Lykera Biomed Sa S100A4 antibodies and therapeutic uses thereof
US9657092B2 (en) 2010-06-14 2017-05-23 Jose Luis Hernandez Miguez S100A4 antibodies and therapeutic uses thereof
WO2015085097A1 (en) 2013-12-05 2015-06-11 The Broad Institute, Inc. Compositions and methods for identifying and treating cachexia or pre-cachexia
EP3633381A2 (en) 2013-12-05 2020-04-08 The Broad Institute, Inc. Compositions and methods for identifying and treating cachexia or pre-cachexia
US11058903B2 (en) 2013-12-05 2021-07-13 The Broad Institute, Inc. Methods for identifying and treating cachexia or pre-cachexia using an inhibitor of rage
EP3733701A1 (en) 2015-03-31 2020-11-04 MedImmune Limited A novel il33 form, mutated forms of il33, antibodies, assays and methods of using the same
WO2016201368A1 (en) 2015-06-10 2016-12-15 The Broad Institute Inc. Antibodies, compounds and screens for identifying and treating cachexia or pre-cachexia
EP3822291A1 (en) 2015-06-10 2021-05-19 The Broad Institute Inc. Antibodies, compounds and screens for identifying and treating cachexia or pre-cachexia
WO2017106196A1 (en) * 2015-12-14 2017-06-22 The Broad Institute, Inc. Compositions and methods for treating cardiac dysfunction
US11111296B2 (en) 2015-12-14 2021-09-07 The Broad Institute, Inc. Compositions and methods for treating cardiac dysfunction
WO2021089559A1 (en) 2019-11-04 2021-05-14 Medimmune Limited Anti il-33 therapeutic agent fpr treating renal disorders
WO2021089563A1 (en) 2019-11-04 2021-05-14 Medimmune Limited Methods of using il-33 antagonists

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