WO2004058184A2 - Anti-ngf antibodies and methods using same - Google Patents

Anti-ngf antibodies and methods using same Download PDF

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Publication number
WO2004058184A2
WO2004058184A2 PCT/US2003/041252 US0341252W WO2004058184A2 WO 2004058184 A2 WO2004058184 A2 WO 2004058184A2 US 0341252 W US0341252 W US 0341252W WO 2004058184 A2 WO2004058184 A2 WO 2004058184A2
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Prior art keywords
antibody
ngf
seq
antibodies
amino acid
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PCT/US2003/041252
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English (en)
French (fr)
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WO2004058184A3 (en
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David L. Shelton
Jaume Pons
Arnon Rosenthal
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Rinat Neuroscience Corp.
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Priority to AU2003299898A priority Critical patent/AU2003299898B2/en
Priority to EA200501043A priority patent/EA011479B1/ru
Priority to MXPA05006854A priority patent/MXPA05006854A/es
Priority to ES03800170T priority patent/ES2382918T3/es
Priority to EP03800170A priority patent/EP1575517B1/en
Priority to DK03800170.7T priority patent/DK1575517T3/da
Priority to KR1020057011944A priority patent/KR101250818B1/ko
Priority to CA2511598A priority patent/CA2511598C/en
Priority to CN2003801099334A priority patent/CN101014364B/zh
Priority to BRPI0317738A priority patent/BRPI0317738B8/pt
Priority to NZ540730A priority patent/NZ540730A/en
Priority to SI200332143T priority patent/SI1575517T1/sl
Priority to JP2005510064A priority patent/JP4134170B2/ja
Priority to EP18195127.8A priority patent/EP3539569A1/en
Priority to AT03800170T priority patent/ATE553128T1/de
Priority to EP10176615.2A priority patent/EP2263692B1/en
Priority to KR1020127025397A priority patent/KR101410692B1/ko
Application filed by Rinat Neuroscience Corp. filed Critical Rinat Neuroscience Corp.
Publication of WO2004058184A2 publication Critical patent/WO2004058184A2/en
Priority to IL169221A priority patent/IL169221A/en
Priority to NO20053583A priority patent/NO336655B1/no
Priority to HK05110620.2A priority patent/HK1076251A1/xx
Publication of WO2004058184A3 publication Critical patent/WO2004058184A3/en
Priority to HK08101386.2A priority patent/HK1110513A1/xx
Priority to AU2011201157A priority patent/AU2011201157C1/en
Priority to IL218087A priority patent/IL218087B/en
Priority to NO20150469A priority patent/NO339595B1/no
Priority to IL263573A priority patent/IL263573A/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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention concerns anti-NGF antibodies (such as anti-NGF antagonist antibodies).
  • the invention further concerns use of such antibodies in the treatment and/or prevention of pain, including post-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain.
  • Nerve growth factor was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons. Smeyne et al, Nature 368:246-249 (1994) and Crowley et al., Cell 76:1001-1011 (1994).
  • NGF up-regulates expression of neuropeptides in sensory neurons (Lindsay and Harmer, Nature 337:362-364 (1989)) and its activity is mediated through two different membrane-bound receptors, the TrkA receptor and the p75 common neurotrophin receptor (sometimes termed "high affinity” and “low affinity” NGF receptors, respectively).
  • TrkA receptor the TrkA receptor
  • p75 common neurotrophin receptor sometimes termed "high affinity” and "low affinity” NGF receptors, respectively.
  • Nerve growth factor was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the developement of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons (Smeyne, et al., Nature 368:246-249 (1994) and Crowley, et al., Cell 76:1001-1011 (1994)).
  • NGF upregulates expression of neuropeptides in sensory neurons (Lindsay, et al., Nature 337:362-364 (1989)), and its activity is mediated through two different membrane-bound receptors, the TrkA tyrosine kinase receptor and the p75 receptor which is structurally related to other members of the tumor necrosis factor receptor family (Chao, et al., Science 232:518-521 (1986)).
  • NGF has been increasingly implicated in processes outside of the nervous system.
  • NGF has been shown to enhance vascular permeability (Otten, et al., Eur J Pharmacol. 106:199-201 (1984)), enhance T- and B-cell immune responses (Otten, et al., Proc. Natl. Acad. Sci. USA 86:10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988); Pearce, et al., J. Physiol.
  • NGF is produced by a number of cell types including mast cells (Leon, et al., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)), B-lymphocytes (Torcia, et al., Cell 85:345-356 (1996), keratinocytes (Di Marco, et al., J. Biol. Chem. 268:22838-22846)), smooth muscle cells (Ueyama, et al., J. Hypertens. 11:1061-1065 (1993)), f ⁇ broblasts (Lindholm, et al, Ewr. J Neurosci.
  • mast cells Leon, et al., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)
  • B-lymphocytes Torcia, et al., Cell 85:345-356 (1996)
  • keratinocytes Di Marco, et al., J. Biol
  • NGF receptors have been found on a variety of cell types outside of the nervous system. For example, TrkA has been found on human monocytes, T- and B-lymphocytes and mast cells.
  • Rodent anti-NGF antagonist antibodies have been reported. See, e.g.,
  • the invention disclosed herein concerns antibodies to nerve growth factor.
  • the invention is a humanized and affinity matured antibody, E3, which specifically binds human and rodent nerve growth factor ("NGF").
  • E3 The amino acid sequences of the heavy chain and light chain variable regions of E3 are shown in Figures 1 A (SEQ ID NO:l) and IB (SEQ ID NO:2), respectively.
  • the CDR portions of antibody E3 (including Chothia and Kabat CDRs) are diagrammatically depicted in Figures IA and IB.
  • the amino acid sequences of E3 heavy and light chains, and of the individual extended CDRs are also shown below (See, "antibody sequences", below).
  • the invention is an antibody comprising a fragment or a region of the antibody E3 (interchangeably termed "E3" herein).
  • the fragment is a light chain of the antibody E3 as shown in Figure IB.
  • the fragment is a heavy chain of the antibody E3 as shown in Figure 1 A.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody E3.
  • the fragment contains one or more complementarity determining regions (CDRs) from a light chain and/or a heavy chain of the antibody E3 as shown in Figure 1 A and IB.
  • CDRs complementarity determining regions
  • the invention is an antibody comprising a light chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894.
  • the invention is an antibody comprising a heavy chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4895.
  • the invention is an antibody comprising (a) a light chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4894 or ATCC No.
  • the invention is an antibody comprising a light chain variable region of a light chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893.
  • the invention is an antibody comprising a heavy chain variable region of a heavy chain that that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA- 4895.
  • the invention is an antibody comprising (a) a light chain variable region of a light chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893, and (b) a heavy chain variable region of a heavy chain that that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4895.
  • the invention is an antibody comprising one or more CDR(s) encoded by (a) a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA- 4894; and/or (b) a heavy chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4895.
  • the antibody comprises the human heavy chain
  • the antibody comprises the human light chain kappa constant region.
  • the antibody comprises a modified constant region, such as a constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
  • the constant region is modified as described in Ear. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.
  • the antibody comprises a human heavy chain IgG2a constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype IgG2a sequence). Eur. J. Immunol. (1999) 29:2613-2624.
  • polypeptides (which may or may not be an antibody) comprising any one or more of the following: a) one or more CDR(s) of antibody E3 shown in Figures IA and IB; b) CDR H3 from the heavy chain of antibody E3 shown in figure IA; c) CDR L3 from the light chain of antibody E3 shown in Figure IB; d) three CDRs from the light chain of antibody E3 shown in Figure IB; e) three CDRs from the heavy chain of antibody E3 shown in Figure IA; and f) three CDRs from the light chain and three CDRs from the heavy chain, of antibody E3 shown in Figures 1 A and IB.
  • the invention further provides polypeptides (which may or may not be an antibody) comprising any one or more of the following: a) one or more (one, two , three, four, five, or six) CDR(s) derived from antibody E3 shown in Figures 1 A and IB; b) a CDR derived from CDR H3 from the heavy chain of antibody E3 shown in Figure IA; and/or c) a CDR derived from CDR L3 from the light chain of antibody E3 shown in Figure IB.
  • the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or “combined” CDRs herein).
  • polypeptides (such as an antibody) bind NGF (such as human NGF).
  • the polypeptides comprise any of the CDF configurations (including combinations, variants, etc.) described herein.
  • the invention provides polypeptides (such as an antibody), which comprise a heavy chain variable region comprising SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is substituted withN, T or S.
  • polypeptides such as an antibody
  • a heavy chain variable region comprising SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is substituted withN, T or S.
  • substituted or “is” in this context or reference to an amino acid refers to choices of amino acid(s) for a given position.
  • substitution, or choice may be the amino acid depicted in a SEQ ID or Figure.
  • the invention provides polypeptides (such as an antibody) which comprise a heavy chain variable region comprising SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V.
  • the invention provides polypeptides (such as an antibody) which comprises a heavy chain variable region comprising SEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is D, N, or G; and wherein Yl 10 is Y, K, S, R or T.
  • the invention provides polypeptides (such as an antibody) which comprise a heavy chain variable region comprising SEQ ID NO:l 1, wherein YlOO is
  • Y, L, or R wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • polypeptides such as an antibody
  • polypeptides which comprise a heavy chain variable region comprising SEQ ID NO: 11, wherein G98 is
  • polypeptides (such as an antibody) which comprise a light chain variable region comprising SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q.
  • polypeptides (such as an antibody) which comprise a light chain variable region comprising SEQ ID NO: 13, wherein 151 is I,
  • T V or A
  • S56 is S or T.
  • polypeptides (such as an antibody) which comprise a light chain variable region comprising SEQ ID NO: 14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.
  • polypeptides (such as an antibody) which comprise a light chain variable region comprising SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the invention provides polypeptides (such as an antibody), which comprise an amino acid sequence shown in SEQ ID NO: 9, wherein 134 is S, L, V A, or I; andN35 is N, T or S.
  • polypeptides such as an antibody which comprise an amino acid sequence shown in SEQ ID NO: 10, wherein M50 is M, I, G,
  • the invention provides polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Yl 10 is Y, K, S, R or T.
  • the invention provides polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO:l 1, wherein YlOO is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • polypeptides such as an antibody
  • the invention provides polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO:l 1, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V, N, D, or T; wherein YlOO is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q.
  • the invention provides polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 13, wherein 151 is I, T, V or A; and S56 is S or T.
  • polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.
  • polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the invention provides polypeptides (such an antibodies, including humanized antibodies) which comprise a heavy chain variable region comprising the CDRl region of SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is N, T or S; the CDR2 region of SEQ ID NO: 10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3 region of SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is D, N, or G; wherein Yl 10 is Y, K, S, R
  • the heavy chain variable region comprises the CDR3 region of SEQ ID NO:l 1, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; wherein Yl 10 is any amino acid.
  • the heavy chain variable region comprises the CDR3 region of SEQ ID NO:ll, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V, N, D, or T; wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • the polypeptide (such as an antibody) further comprises an antibody light chain variable region.
  • the invention provides polypeptides (such as an antibody) which comprise a light chain variable region comprising the CDRl region of SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; the CDR2 region of SEQ ID NO:13, wherein 151 is I, T, V or A; and S56 is S or T; and the CDR3 region of SEQ ID NO: 14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.
  • polypeptides such as an antibody
  • the light chain variable region comprises the CDR3 region of SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the polypeptide (such as an antibody) further comprises an antibody heavy chain.
  • polypeptides (such as an antibody) which comprise (a) a heavy chain variable region comprising the CDRl region of SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is N, T or S; the CDR2 region of SEQ ID NO: 10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3 region of SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; wherein Yl 10 is Y, K, S,
  • the light chain variable region comprises the CDR3 region of SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the heavy chain variable region comprises the CDR3 region of SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; wherein Yl 10 is any amino acid.
  • the heavy chain variable region comprises the CDR3 region of SEQ ID NO: 11 , wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V, N, D, or T; wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F 108 is F or W; wherein D 109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • the polypeptide further comprises an antibody light chain.
  • polypeptides such an antibody, including a humanized antibody
  • polypeptides which comprise an amino acid sequence shown in SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is N, T or S; an amino acid sequence shown in SEQ ID NO: 10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and an amino acid sequence shown in SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is D, N, or G; wherein Yl 10 is Y, K, S, R or T.
  • the polypeptide comprises an amino acid sequence shown in SEQ ID NO:l 1, wherein YlOO is Y, L, or R; and wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • the polypeptide comprises an amino acid sequence shown in SEQ ID NO:l 1, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V, N, D, or T; wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F 108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • the polypeptide (such as an antibody) further comprises an antibody light chain variable region.
  • the invention provides polypeptides (such as an antibody) which comprise an amino acid sequence shown in SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; an amino acid sequence shown in SEQ ID NO:13, wherein 151 is I, T, V or A; and S56 is S or T; and an amino acid sequence shown in SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.
  • polypeptides such as an antibody
  • thepolypeptide comprises an amino acid sequence shown in SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the polypeptide (such as an antibody) further comprises an antibody heavy chain variable region.
  • polypeptides (such as an antibody) which comprise (a) an amino acid sequence shown in SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is N, T or S; an amino acid sequence shown in SEQ ID NO: 10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and an amino acid sequence shown in SEQ ID NO:l 1, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F 108 is F or W; wherein D 109 is D, N, or G; and wherein Yl 10 is Y, K, S, R or T; and
  • the polypeptide comprises an amino acid sequence shown in SEQ ID NO: 14, wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.
  • the polypeptide comprises an amino acid sequence shown in SEQ ID NO:l 1, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; wherein Yl 10 is any amino acid.
  • the polypeptide comprises an amino acid sequence shown in SEQ ID NO:l 1, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V, N, D, or T; wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherem T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Yl 10 is any amino acid.
  • the polypeptide further comprises an antibody light chain variable region.
  • polypeptide comprising a heavy chain variable region comprising: (a) a CDRl region of SEQ ID NO:9, wherein 134 is S, L, V A, or I; and N35 is substituted with N, T or S; (b) a CDR2 region of SEQ ID NO: 10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and (c) a CDR3 region of SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein SI 05 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Y
  • the invention provides polypeptides (such as antibodies) comprising a light chain variable region comprising: (a) a CDRl region of SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; (b) a CDR2 region of SEQ ID NO: 13, wherein 151 is I, T, V or A; and S56 is S or T; and (c) a CDR3 region of SEQ ID NO: 14, wherein K92 is K, H, R, or S; and wherein Y96 is Y or R; wherein the antibody binds NGF.
  • polypeptides comprising a light chain variable region comprising: (a) a CDRl region of SEQ ID NO: 12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; (b) a CDR2 region of SEQ ID NO: 13, wherein 151 is I, T, V
  • polypeptides comprising (a) a heavy chain variable region comprising: (i) a CDRl region of SEQ ID NO:9, wherein 134 is substituted with S, L, V A, or I; and N35 is substituted with N, T or S; (ii) a CDR2 region of SEQ ID NO: 10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and (iii) a CDR3 region of SEQ ID NO: 11, wherein YlOO is Y, L, or R; wherein YlOl is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D 109 is D,
  • polynucleotides (such as an antibody) bind NGF
  • variable region numbering used herein is sequential numbering.
  • a number of antibody numbering systems exist (such as Kabat and Chothia numbering), and how to convert sequential numbering into another numbering system, such as Kabat numbering or Chothia numbering.
  • the invention provides a polypeptide (such as an antibody) comprising an amino acid sequence (such as a CDR3 sequence) selected from SEQ ID NO:46 or 50.
  • the polypeptide further comprises one or more of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and 8.
  • the polypeptide further comprises one of more of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.
  • the invention provides a polypeptide (such as an antibody) comprising an amino acid sequence (such as a CDR region, such as a CDRHl and/or CDR H2 region) selected from (a) SEQ ID NOS:28 and/or 29; (b) SEQ ID NOS:30 and/or 31; (c) SEQ ID NOS:32 and/or 33; (d) SEQ ID NOS:34 and/or 35; (e) SEQ ID NOS:36 and/or 37; (f) SEQ ID NOS:38 and/or 39; and (g) SEQ ID NOS:40 and 41.
  • an amino acid sequence such as a CDR region, such as a CDRHl and/or CDR H2 region
  • the polypeptide comprises an amino acid sequence (such as a CDR HI region) selected from SEQ ID NOS:28, 30, 32, 34, 36, 38, and 40. In some embodiments, the polypeptide comprises an amino acid sequence (such as a CDR H2 region) selected from SEQ ID NOS:29, 31, 33, 35, 37, 39 and 41. In still other embodiments, the polypeptide further comprises one or more of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still other embodiments, the polypeptide further comprises one of more of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.
  • the invention provides a polypeptide (such as an antibody) comprising an amino acid sequence (such as a CDR region, such as a CDRL1 and/or CDR L2 region) selected from (a) SEQ ID NOS:18 and/or 19; (b) SEQ ID NOS:20 and/or 21; and (c) SEQ ID NOS:22 and/or 23.
  • the polypeptide comprises an amino acid sequence (such as a CDR LI region) selected from SEQ ID NOS:18, 20, and 22.
  • the polypeptide comprises an amino acid sequence (such as a CDR L2 region) selected from SEQ ID NOS: 19, 21, and 23.
  • polypeptide further comprises one or more of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, 8. In still other embodiments, the polypeptide further comprises one of more of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.
  • the invention provides a polypeptide (such as an antibody) comprising an amino acid sequence (such as a CDR region, such as a CDRL3 and/or CDR H3 region) selected from (a) SEQ ID NOS:51 and/or 52; (b) SEQ ID NOS:55 and/or 56; (c) SEQ ID NOS:57 and/or 58; (c) SEQ ID NOS:59 and/or 60; (d) SEQ ID NOS:61 and/or 62; (e) SEQ ID NOS:63 and/or 64.
  • an amino acid sequence such as a CDR region, such as a CDRL3 and/or CDR H3 region
  • the polypeptide comprises an amino acid sequence (such as a CDR L3 region) selected from SEQ ID NOS:51, 55, 57, 59, 61, and 63. In some embodiments, the polypeptide comprises an amino acid sequence (such as a CDR H3 region) selected from SEQ ID NOS:52, 56, 58, 60, 62, and 64. In still other embodiments, the polypeptide further comprises an amino acid sequence shown in one or more of SEQ ID NOS: 18, 19, 30 and 31. In still other embodiments, the polypeptide further comprises one or more of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and 8.
  • the polypeptide further comprises one of more of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.
  • the invention provides a polypeptide (such as an antibody) comprising one or more of an amino acid sequence (such as a CDR region) shown in SEQ ID NOS:61, 63, 18, 19, 30 and 31.
  • the invention provides an anti-NGF antibody (such as an antagonist antibody) that binds NGF (such as human NGF) with a high affinity.
  • high affinity is (a) binding NGF with a K D of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or less), and/or a ko f of slower than about 6x10 "5 s "1 ); and/or (b) inhibiting (reducing, and/or blocking) human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or
  • the invention provides polypeptides (such as an antibody), wherein the polypeptides (a) bind NGF (such as human NGF) with a KD of less than about 2 nM (such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, lOOpM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or less), and/or a k 0ff of slower than about 6xl0 "5 s "1 ); and/or (b) inhibit human NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or less; and/or (c) inhibit human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an
  • the polypeptides (a) bind NGF with a K D of less than about 2 nM; and/or (b) inhibit human NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 of about 100 pM or less, wherein the IC50 is measured in the presence of about 15 pM NGF; and/or (c) inhibit human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 of about 10 pM or less, wherein the IC50 is measured in the presence of about 1.5 pM of NGF, wherein the IC50 is measured in the presence of about 15 pM NGF.
  • the polypeptides (a) bind NGF with a K D of less than about 100 pM; and/or (b) inhibit human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 of about 20 pM or less, wherein the IC50 is measured in the presence of about 15 pM NGF; and/or (c) inhibit human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 of about 2 pM or less, wherein the IC50 is measured in the presence of about 1.5 pM of NGF.
  • polypeptide embodiments consisting of the identical amino acid sequence to an amino acid sequence of mouse monoclonal antibody, 911.
  • the extended CDR sequences of Mab 911 are shown in Figures 1 A and IB, and in SEQ ID NOS:9-14.
  • the invention provides any of the above polypeptides or antibodies, further wherein the polypeptide (such as an antibody) is isolated.
  • the polypeptide (such as an antibody) is substantially purified.
  • the polypeptide (such as an antibody) is affinity matured.
  • the antibody is an antagonist antibody.
  • the polypeptide (such as an antibody) comprises human framework sequences.
  • the polypeptide (such as an antibody) comprises one or more non-human framework residues.
  • the polypeptide (such as an antibody) binds NGF (such as human NGF) with a K D of 2nM or less.
  • the polypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, or more) human amino acid substitutions relative to a non-human amino acid sequence (such as a variable region sequence, such as a CDR sequence, such as a framework sequence).
  • the polypeptide comprises at least 1, at least 2, or more such as at least 3, 4, 5, 6, or more amino acid substitutions relative to a parent polypeptide amino acid sequence (such as an antibody 911 amino acid sequence, such as any one or more of SED ID NOs 9- 14).
  • the binding affinity of the antibody has been altered (in some embodiments, increased) relative to a parent antibody (such as Mab 911) affinity.
  • the binding affinity of the antibody is lower than the binding affinity of trkA receptor for NGF (such as human NGF).
  • the polypeptides may be antibodies.
  • the antibodies are human antibodies. In other embodiments, the antibodies are humanized antibodies.
  • the antibodies are monoclonal antibodies. In some embodiments, the antibody is an affinity matured antibody.
  • the invention provides polynucleotides (including isolated polynucleotide) comprising polynucleotides encoding any of the embodiments above. [0059] In another aspect, the invention provides an isolated polynucleotide comprising a polynucleotide encoding a fragment or a region of the antibody E3 (interchangeably termed "E3" herein). In one embodiment, the fragment is a light chain of the antibody E3 as shown in Figure IB. In another embodiment, the fragment is a heavy chain of the antibody E3 as shown in Figure 1 A.
  • the fragment contains one or more variable regions from a light chain and/or a heavy chain of the antibody E3. In yet another embodiment, the fragment contains one or more complementarity determining regions (CDRs) from a light chain and/or a heavy chain of the antibody E3 as shown in Figures IA and IB.
  • CDRs complementarity determining regions
  • the invention is an isolated polynucleotide comprising a polynucleotide that encodes for antibody E3.
  • the polynucleotide comprises either or both of the polynucleotide shown in Figures 2 and 3.
  • the invention is an isolated polynucleotide that encodes for an E3 light chain with a deposit number of ATCC No. PTA-4893 or ATCC No. PTA- 4894.
  • the invention is an isolated polynucleotide that encodes for an E3 heavy chain with a deposit number of ATCC No. PTA-4895.
  • the invention is an isolated polynucleotide comprising (a) a variable region encoded in the polynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894 and (b) a variable region encoded in the polynucleotide with a deposit number of ATCC No. PTA- 4895.
  • the invention is an isolated polynucleotide comprising (a) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894; and/or (b) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA-4895.
  • the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) or polypeptides described herein.
  • the invention provides vectors (including expression and cloning vectors) and host cells comprising any of the polynucleotide disclosed herein.
  • vectors including expression and cloning vectors
  • host cells comprising any of the polynucleotide disclosed herein.
  • polynucleotide embodiments consisting of the identical polynucleotide sequence to a polynucleotide sequence of mouse monoclonal antibody, 911.
  • the extended CDR sequences of Mab 911 are shown in Figures 1 A and IB, and in SEQ ID NOS:9-14.
  • the invention is a host cell comprising a polynucleotide encoding E3 light chain and a polynucleotide encoding E3 heavy chain, wherein the polynucleotide(s) encoding E3 light chain has a deposit number of ATCC No. PTA-4893 and/or ATCC No. PTA-4894, and the polynucleotide encoding E3 heavy chain has a deposit number of ATCC No. PTA-4895.
  • the host cell comprises polynucleotide comprising (a) a variable region encoded in the polynucleotide with a deposit number of ATCC No.
  • the host cell comprises a polynucleotide encoding (a) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA-4893 or PTA- 4894; and/or (b) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA-4895.
  • the host cell is a mammalian cell.
  • the invention is a complex of NGF bound by antibody E3.
  • the complex is isolated. In another aspect, the complex is substantially purified.
  • the invention is a complex of NGF bound by any of the antibodies or polypeptides described herein.
  • the complex is isolated.
  • the complex is substantially purified.
  • the invention is a pharmaceutical composition comprising any of the polypeptides (including antibodies such as antibody E3) or polynucleotides described herein, such as pharmaceutical compositions comprising the antibody E3 or an antibody comprising a fragment of the antibody E3, and a pharmaceutically acceptable excipient.
  • the invention is a method of generating antibody E3 comprising preparing a host cell comprising an expression vector that encodes for antibody E3; culturing the host cell or progeny thereof under conditions that allow production of antibody E3; and purifying the antibody E3.
  • the expression vector comprises one or both of the polynucleotide sequences shown in Figures 2 and 3.
  • the invention is a method of generating antibody E3 comprising expressing a polynucleotide encoding E3 light chain and a polynucleotide encoding E3 heavy chain in a suitable cell, wherein the polynucleotide encoding E3 light chain has a deposit number of ATCC No.
  • PTA-4893 and/or ATCC No. PTA-4894, and the polynucleotide encoding E3 heavy chain has a deposit number of ATCC No. PTA-4895; generally followed by recovering and/or isolating the antibody.
  • the invention provides methods of generating any of the polypeptides (such as antibodies) described herein by expressing one or more polynucleotides encoding the antibody (which may be separately expressed as a single light or heavy chain, or both a light and a heavy chain may be expressed from one vector) in a suitable cell, generally followed by recovering and/or isolating the antibody or polypeptides of interest.
  • the invention is a method of antagonizing NGF (such as human NGF) biological activity using any of the polypeptides (including antibodies such as antibody E3) disclosed herein.
  • the method comprises contacting human nerve growth factor with any of the polypeptides (including antibody E3) described herein, whereby NGF activity (such as human nerve growth factor activity) is antagonized, reduced, blocked, or suppressed.
  • the invention is a method of detecting NGF using any of the polypeptides (including antibodies, such as the antibody E3) described herein.
  • the presence of NGF is detected by detecting a complex between NGF and any of the polypeptides described herein (such as antibody E3).
  • detection includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.
  • the invention is a method of treating pain by administering an effective amount of a composition comprising the antibody E3 or any of the polypeptide (including antibody) or polynucleotide embodiments described herein.
  • the pain is post-surgical pain.
  • the invention is a method for preventing or treating rheumatoid arthritis pain in an individual by administering an effective amount of anti-NGF antagonist antibody to the individual. It has been shown in accordance with the invention that an anti-NGF antagonist antibody is capable of inhibiting or blocking the pain associated with rheumatoid arthritis.
  • the pain is alleviated within about 24 hours after administering the anti-NGF antagonist antibody. In some embodiments, the pain is alleviated within about 4 days after administering the anti-NGF antagonist antibody. In some embodiments, the pain is alleviated before observing or in the absence of an indication of improvement of the inflammatory condition in the individual.
  • the invention provides methods for reducing incidence of rheumatoid arthritis pain, ameliorating rheumatoid arthritis pain, suppressing rheumatoid arthritis pain, palliating rheumatoid arthritis pain, and/or delaying the onset, development, or progression of rheumatoid arthritis pain in an individual, said method comprising administering an effective amount of anti-NGF antagonist antibody to the individual.
  • the invention is a method for preventing or treating osteoarthritis pain in an individual by administering an effective amount of anti-NGF antagonist antibody to the individual.
  • the invention provides methods for treating inflammatory cachexia (weight loss) associated with rheumatoid arthritis in an individual comprising administering an effective amount of an anti-NGF antagonist antibody.
  • the invention provides methods for reducing incidence of osteoarthritis pain, ameliorating osteoarthritis pain, suppressing osteoarthritis pain, palliating osteoarthritis pain, and/or delaying the onset, development, or progression of osteoarthritis pain in an individual, said method comprising administering an effective amount of anti-NGF antagonist antibody to the individual.
  • kits and compositions comprising any one or more of the compositions described herein. These kits, generally in suitable packaging and provided with appropriate instructions, are useful for any of the methods described herein.
  • the invention also provides any of the compositions and kits described for any use described herein whether in the context of use as medicament and/or use for manufacture of a medicament.
  • FIGURE 1 A shows the amino acid sequence of the heavy chain variable region of the E3 antibody (labeled “6” and “5 + affinity maturation H3).
  • the Chothia CDRs and Kabat CDRs are depicted by underlined text and bold and italicized text, respectively.
  • Figure 1 A also shows the alignment of the following heavy chain variable region amino acid sequences; (2) VH4-59 human germline acceptor sequence (labeled “VH4-59” or “2”); (3) the acceptor sequences grafted with the extended CDRs of the mouse antibody 911 (labeled “CDR grafted” or “3”); (4) the CDR grafted acceptor sequences including the V71K substitution (labeled ""3+one framework mutation” or "4"); (5) the clone containing affinity matured CDRs HI and H2 (labeled "5" or "4+ affinity maturation HI, H2"); and antibody E3 (as described above).
  • FIGURE IB shows the amino acid sequence of the light chain variable region of the E3 antibody (labeled "5" or "4 + affinity maturation L3).
  • the Chothia CDRs and Kabat CDRs are depicted by underlined text and bold and italicized text, respectively.
  • FIGURE 2 shows a polynucleotide comprising a polynucleotide sequence encoding the heavy chain variable region of antibody E3.
  • FIGURE 3 shows a polynucleotide comprising a polynucleotide sequence encoding the light chain variable region of antibody E3.
  • FIGURE 4 is a graph depicting NGF-dependent survival of E13.5 neurons in the presence of varying concentration of human and rat NGF.
  • the X axis corresponds to NGF concentration (ng/ml) and the Y axis corresponds to counted neurons.
  • FIGURE 5 is a graph comparing the NGF blocking effect of various Fabs in the presence of either 0.04 ng/ml of human NGF (approximately 1.5 pM; shown in lower panel) or 0.4 ng/ml human NGF (approximately 15 pM; shown in upper panel).
  • Fab E3 strongly blocked human NGF-dependent trigeminal neuron survival, with an IC50 of approximately 21 pM in the presence of 15 pM human NGF, and an IC50 of approximately 1.2 pM in the presence of 1.5 pM human NGF.
  • Fabs 3C and H19-L129 also strongly blocked human NGF-dependent trigeminal neuron survival.
  • FIGURE 6 is a graph comparing the NGF blocking effect of various Fabs in the presence of either 0.04 ng/ml of rat NGF (approximately 1.5 pM; shown in lower panel) or 0.4 ng/ml rat NGF (approximately 15 pM; shown in upper panel).
  • Fab E3 strongly blocked human NGF-dependent trigeminal neuron survival, with an IC50 of approximately 31.6 pM in the presence of 15 pM rat NGF, and an IC50 of approximately 1.3 pM in the presence of 1.5 pM rat NGF.
  • Fabs 3 ' C and HI 9-L129 also strongly blocked rat NGF-dependent trigeminal neuron survival.
  • 1.5 pM of NGF was around the IC50, while 15 pM represented a saturating concentration of NGF.
  • the X axis corresponds to antibody concentration (nM) and the Y axis corresponds to counted neurons.
  • FIGURE 7 is a graph depicting resting pain assessed 24 hours after surgery and showing that treatment with 0.02 mg/kg, 0.1 mg/kg, 0.6 mg/kg, or 1 mg/kg of anti- NGF antibody E3 reduced pain. "*" indicates a statistically significant difference (p ⁇ 0.5) from the negative control.
  • FIGURE 8 is a graph depicting resting pain assessed 24 hours after surgery and showing that treatment with 0.5 mg/kg of anti-NGF antibody E3 significantly (p ⁇ 0.005) reduced resting pain when injected two hours after surgery.
  • FIGURE 9 is a graph showing the results of BIAcore analysis of the binding affinity to human NGF of mouse antibody 911 (Fab).
  • FIGURE 10 is a graph showing the results of BIAcore analysis of the binding affinity to human NGF of antibody E3 (Fab) (referred to as “3E Fab").
  • E3 bound human NGF with a KD of approximately 0.07 nM (and with a kon of about 6.0 x 10 5 M _1 s " and a k of of about 4.2x10 "5 s "1 ).
  • FIGURE 11 is a graph depicting that antibody E3 blocks the interaction of
  • NGF with its receptors trkA and p75, as assessed by percent binding detected between NGF and trkA (shown in black circles) and NGF and p75 (shown as hollow squares).
  • the X axis corresponds to concentration of antibody 3E (Fab) and the Y axis corresponds to NGF binding (percent maximum RU).
  • Increased concentrations of Fab E3 blocked the interaction of NGF with both p75 and trkA, as shown by decreased signal (measured in RU).
  • antibody E3 (Fab) concentration equaled NGF concentration, no NGF binding was observed (as shown by a signal of zero).
  • FIGURE 12 is a graph depicting the human NGF blocking ability of full antibody E3 and Fab E3. Survival of E13.5 mouse trigeminal neurons in the presence of human NGF and various concentrations of Fab E3 and antibody E3 was assessed. The X axis corresponds to NGF binding sites (nM) and the Y axis corresponds to normalized count of trigeminal (TG) neurons. Full antibody E3 and Fab 3E showed similar levels of inhibition of NGF-dependent survival of trigeminal neurons when the concentration of whole antibody and Fab were normalized to the number of NGF binding sites (Fab has one binding site and whole antibody has two binding sites).
  • FIGURE 13 is a graph depicting the ability of various concentrations (20,
  • FIGURE 14 is a graph depicting that anti-NGF antagonist antibody E3
  • FIGURE 15 is a graph depicting that anti-NGF antagonist antibody E3
  • FIGURE 16 is a graph depicting that anti-NGF antagonist antibody E3 or
  • FIGURE 17 is a graph depicting that anti-NGF antagonist antibody E3 or
  • Fab E3 did not inhibit survival of El 7 nodose neurons promoted by BDNF, NT4/5 or LIF.
  • Various concentrations (200 nM, 20 nM, 2nM) of Fab E3 (termed “3E in the figure"), or Fab 911 were tested in the presence of no added neurotrophins (termed “control”), 400 pM BDNF (termed “BDNF-400pM), 400 pM NT4/5 (termed “NT4/5-400pM), or 2.5 nM LIF (termed "LIP-2.5 nM).
  • FIGURE 18 is a graph demonstrating nociceptive response in arthritic rats
  • FIGURE 19 is a graph demonstrating effects of anti-NGF antibodies on body weight in arthritis in rats (rheumatoid arthritis model) after administration of anti- NGF antibodies on D14 and D19.
  • FIGURE 20 is a graph demonstrating nociceptive response in arthritic rats
  • E3 (0.003 mg/kg, 0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg) on D14 and D18.
  • Vocalization intensity values are expressed in mV as means ⁇ s.e.m.
  • FIGURE 21 is a graph demonstrating effects of anti-NGF antibody E3 on percentage of weight on Day 14 (normalized to Day 14) in arthritic rats (rheumatoid arthritis model) after administration of different doses of anti-NGF antibody E3 (0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg) on D14 and D18.
  • FIGURE 22 is a graph demonstrating effects of anti-NGF antibody E3 on weight loss in arthritic rats (rheumatoid arthritis model) after administration of different doses of anti-NGF antibody E3 (0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg) on D14 and D18.
  • FIGURE 23 depicts the E3 heavy chain variable region amino acid sequence (Fig. 23A) and light chain variable region amino acid sequence (Fig. 23B), as numbered using sequential numbering, Kabat numbering, and Chothia numbering.
  • the invention disclosed herein provides anti-NGF antagonist antibodies that bind NGF (such as human NGF) with high affinity.
  • the invention further provides antibodies and polypeptides derived from E3 that bind NGF, and methods of making and using these antibodies.
  • the invention provides a humanized antibody, E3, which binds to nerve growth factor ("NGF"), and methods of making and using this antibody.
  • the invention also provides E3 polypeptides (including antibodies) that bind NGF, and polynucleotides encoding E3 antibody and/or polypeptide.
  • the invention disclosed herein also provides methods for preventing and/or treating rheumatoid arthritis pain in an individual by administration of a therapeutically effective amount of an anti-NGF antagonist antibody.
  • the invention disclosed herein also provides methods for preventing and/or treating osteoarthritis pain in an individual by administration of a therapeutically effective amount of an anti-NGF antagonist antibody.
  • the invention also provides methods for adjusting the affinity of an antibody and methods for characterizing a CDR region.
  • an "antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known
  • Fv is an antibody fragment that contains a complete antigen-recognition and -binding site.
  • this region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association.
  • one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a dimeric structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding specificity on the surface of the NH-NL dimer.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge regions.
  • a "monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an antigen.
  • a population of monoclonal antibodies is highly specific, being directed against a single antigenic site.
  • the term "monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • human antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art or disclosed herein.
  • This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide.
  • One such example is an antibody comprising murine light chain and human heavy chain polypeptides.
  • Human antibodies can be produced using various techniques known in the art.
  • the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al, 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581).
  • Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos.
  • the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J Immunol, 147 (l):86-95; and U.S. Patent No. 5,750,373.
  • Chimeric antibodies refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chains is homologous to corresponding sequences in another.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another.
  • the variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non human host organisms in combination with constant regions derived from, for example, human cell preparations.
  • variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source.
  • effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region.
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g.
  • variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95% sequence identity therewith.
  • ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • NK natural killer
  • macrophages e.g. NK cells, neutrophils, and macrophages
  • ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.
  • Fc receptor and “FcR” describe a receptor that binds to the
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an "inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev.
  • FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol, 117:587; and Kim et al., 1994, J. Immunol, 24:249).
  • Complement dependent cytotoxicity and “CDC” refer to the lysing of a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.
  • E3 As used herein, the terms “E3”, “3E”, and “antibody E3” are used interchangeably to refer to an antibody comprising the amino acid sequence of the heavy chain and light chain variable regions shown in Figures IA (SEQ ID NO:l) and IB (SEQ ID NO:2), respectively.
  • the CDR portions of antibody E3 (including Chothia and Kabat CDRs) are diagrammatically depicted in Figures 1 A and IB.
  • Figures 2 and 3 show polynucleotides encoding heavy and light chains, respectively, comprising the heavy and light chain variable regions shown in Figures 1 A and IB, respectively. The generation and characterization of E3 is described in the Examples.
  • E3 refers to immunoglobulin encoded by (a) a polynucleotide encoding E3 light chain that has a deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894, and (b) a polynucleotide encoding E3 heavy chain that has a deposit number of ATCC No. PTA-4895.
  • immunospecific binding of antibodies refers to the antigen specific binding interaction that occurs between the antigen-combining site of an antibody and the specific antigen recognized by that antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay, and does not react detectably with unrelated proteins).
  • An epitope that "specifically binds", or “preferentially binds” (used interchangeably herein) to an antibody or a polypeptide is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art.
  • a molecule is said to exhibit "specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically or preferentially binds to an NGF epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other NGF epitopes or non-NGF epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.
  • polypeptide oligopeptide
  • peptide protein
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non- nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2' ⁇ O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, ⁇ -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR 2 ("amidate"), P(O)R, P(O)OR', CO or CH 2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions.
  • the CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • CDRs complementarity determining regions
  • a CDR may refer to CDRs defined by either approach or by a combination of both approaches.
  • a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
  • nerve growth factor and “NGF” refers to nerve growth factor and variants thereof that retain at least part of the biological activity of NGF.
  • NGF includes all mammalian species of native sequence NGF, including human, canine, feline, equine, or bovine.
  • NGF receptor refers to a polypeptide that is bound by or activated by
  • NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including, but are not limited to, human, canine, feline, equine, primate, or bovine.
  • an "anti-NGF antagonist antibody” (interchangeably termed
  • anti-NGF antibody refers to an antibody which is able to bind to NGF and inhibit NGF biological activity and/or downstream pathway(s) mediated by NGF signaling.
  • An anti- NGF antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding and/or elicitation of a cellular response to NGF.
  • an anti-NGF antagonist antibody encompass all the previously identified terms, titles, and functional states and characteristics whereby the NGF itself, an NGF biological activity (including but not limited to its ability to ability to mediate any aspect of post- surgical pain), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti-NGF antagonist antibody binds NGF and prevent NGF dimerization and/or binding to an NGF receptor (such as p75 and/or trkA).
  • an anti-NGF antibody binds NGF and prevents trkA receptor dimerization and/or trkA autophosphorylation. Examples of anti-NGF antagonist antibodies are provided herein.
  • Biological activity of NGF generally refers to the ability to bind NGF receptors and/or activate NGF receptor signaling pathways.
  • a biological activity includes any one or more of the following: the ability to bind an NGF receptor (such as p75 and/or trkA); the ability to promote trkA receptor dimerization and/or autophosphorylation; the ability to activate an NGF receptor signaling pathway; the ability to promote cell differentiation, proliferation, survival, growth and other changes in cell physiology, including (in the case of neurons, including peripheral and central neuron) change in neuronal morphology, synaptogenesis, synaptic function, neurotransmitter and/or neuropeptide release and regeneration following damage; the ability to promote survival of mouse E13.5 trigeminal neurons; and the ability to mediate pain, including post-surgical pain.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), more preferably at least 90 % pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure.
  • a "host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: improvement or alleviation of any aspect of pain, including acute, chronic, inflammatory, neuropathic, post-surgical pain, rheumatoid arthritis pain, or osteoarthritis pain.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: including lessening severity, alleviation of one or more symptoms associated with pain including any aspect of pain (such as shortening duration of pain, reduction of pain sensitivity or sensation).
  • an "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results including clinical results such as alleviation or reduction in pain sensation.
  • An effective amount can be administered in one or more administrations.
  • an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to treat, ameliorate, reduce the intensity of and/or prevent pain, including post-surgical pain, rheumatoid arthritis pain, and/or osteoarthritis pain.
  • the "effective amount” may reduce pain at rest (resting pain) or mechanically-induced pain (including pain following movement), or both, and it may be administered before, during or after an incision, cut, tear or injury and/or before, during or after painful stimulus.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • Reducing incidence of pain means any of reducing severity (which can include reducing need for and/or amount of (e.g., exposure to) other drugs and/or therapies generally used for this conditions, including, for example, opiates), duration, and/or frequency (including, for example, delaying or increasing time to post-surgical pain in an individual).
  • individuals may vary in terms of their response to treatment, and, as such, for example, a "method of reducing incidence of rheumatoid arthritis pain or osteoarthritis pain in an individual" reflects administering the anti-NGF antagonist antibody based on a reasonable expectation that such administration may likely cause such a reduction in incidence in that particular individual.
  • “Ameliorating" a pain or one or more symptoms of a pain means a lessening or improvement of one or , more symptoms of a pain as compared to not administering an anti-NGF antagonist antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.
  • “Palliating" a pain or one or more symptoms of a pain means lessening the extent of one or more undesirable clinical manifestations of post-surgical pain in an individual or population of individuals treated with an anti-NGF antagonist antibody in accordance with the invention.
  • “delaying" the development of pain means to defer, hinder, slow, retard, stabilize, and/or postpone progression of pain, such as post-surgical pain, rheumatoid arthritis pain, or osteoarthritis pain.
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop pain.
  • a method that "delays" development of the symptom is a method that reduces probability of developing the symptom in a given time frame and/or reduces, extent of the symptoms in a given time frame, when compared to not using the method.
  • Pain refers to pain of any etiology, including acute and chronic pain, and any pain with an inflammatory component.
  • pain include post-surgical pain, post-operative pain (including dental pain), migraine, headache and trigeminal neuralgia, pain associated with burn, wound or kidney stone, pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with musculo-skeletal disorders such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism and peri-articular disorders, and pain associated with cancer (including "break-through pain” and pain associated with terminal cancer), peripheral neuropathy and post-herpetic neuralgia.
  • pain with an inflammatory component include rheumatic pain,
  • Post-surgical pain refers to pain arising or resulting from an external trauma such as a cut, puncture, incision, tear, or wound into tissue of an individual (including that that arises from all surgical procedures, whether invasive or non-invasive).
  • post-surgical pain does not include pain that occurs (arises or originates) without an external physical trauma.
  • post-surgical pain is internal or external (including peripheral) pain, and the wound, cut, trauma, tear or incision may occur accidentally (as with a traumatic wound) or deliberately (as with a surgical incision).
  • pain includes nociception and the sensation of pain, and pain can be assessed objectively and subjectively, using pain scores and other methods well-known in the art.
  • Post-surgical pain includes allodynia (i.e., increased response to a normally non-noxious stimulus) and hyperalgesia (i.e., increased response to a normally noxious or unpleasant stimulus), which can in turn, be thermal or mechanical (tactile) in nature.
  • the pain is characterized by thermal sensitivity, mechanical sensitivity and/or resting pain.
  • the post-surgical pain comprises mechanically-induced pain or resting pain. In other embodiments, the post-surgical pain comprises resting pain.
  • a "biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes.
  • the term "biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • An "individual” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats.
  • "vector” means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • expression control sequence means a nucleic acid sequence that directs transcription of a nucleic acid.
  • An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
  • pharmaceutically acceptable carrier includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.
  • K of is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • K d is intended to refer to the dissociation constant of an antibody-antigen interaction.
  • compositions including pharmaceutical compositions, comprising an E3 antibody or polypeptide; and polynucleotides comprising sequences encoding an E3 antibody or polypeptide.
  • compositions comprise one or more antibodies or polypeptides (which may or may not be an antibody) that bind to NGF, and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to NGF.
  • suitable excipients such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
  • the invention also encompasses isolated antibody, polypeptide and polynucleotide embodiments.
  • the invention also encompasses substantially pure antibody, polypeptide and polynucleotide embodiments.
  • the E3 antibody and related antibodies also exhibit a strong capacity to antagonize human NGF, as assessed by in vitro assays (see Examples 2 and 3).
  • antibody E3 antagonizes the NGF-dependent survival of mouse El 3 trigeminal neurons at an IC50 of about 21 pM in the presence of 15 pM of human NGF, and about 1.2 pM in the presence of 1.5 pM of human NGF.
  • the antibodies and polypeptides of the invention are further identified and characterized by: (h) high affinity binding to human NGF with low dissociation kinetics (in some embodiments, with a K D of less than about 2 nM, and/or a koff of slower than about 6x10-5 s-1) and/or (i) ability to inhibit (block) NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 of about 100 pM or less at about 15 pM of NGF (in some embodiments, human NGF) and/or an IC50 of about 20 pM or less at about 1.5 pM of NGF.
  • the antibody binds human NGF, and does not significantly bind an NGF from another vertebrate species (in some embodiment, mammalian). In some embodiments, the antibody binds human NGF as well as one or more NGF from another vertebrate species (in some embodiments, mammalian). In still other embodiments, the antibody binds NGF and does not significantly cross-react with other neurotrophins (such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In some embodiments, the antibody binds NGF as well as at least one other neurotrophin.
  • other neurotrophins such as the related neurotrophins, NT3, NT4/5, and/or BDNF.
  • the antibody binds to a mammalian species of NGF, such as horse or dog, but does not significantly bind to NGF from anther mammalian species.
  • the invention is an antibody comprising a light chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894.
  • the invention is an antibody comprising a heavy chain that is encoded by a polynucleotide that is produced by a host cell with a deposit number of ATCC No. PTA-4895.
  • the present invention also encompasses various formulations of E3 and equivalent antibody fragments (e.g., Fab, Fab', F(ab') 2 , Fv, Fc, etc.), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of E3 that comprises an antigen (NGF) recognition site of the required specificity.
  • equivalent antibodies of E3, including antibody and polypeptide fragments (which may or may not be antibodies) of E3, and polypeptides comprising polypeptide fragments of E3 are identified and characterized by any (one or more) of the criteria described above.
  • the invention provides any of the following, or compositions
  • polypeptide embodiments consisting of the identical amino acid sequence to an amino acid sequence of mouse monoclonal antibody, 911.
  • the extended CDR sequences of Mab 911 are shown in Figures 1 A and IB, and in SEQ ID NOS.9-14.
  • the CDR portions of antibody E3 are diagrammatically depicted in Figures IA and IB, and consist of the following amino acid sequences: (a) heavy chain CDR 1 ("CDR HI”) GFSLIGYDLN (SEQ ID NO:3); (b) heavy chain CDR 2 ("CDR H2") IIWGDGTTDYNSANKS (SEQ ID ⁇ O:4); (c) heavy chain CDR 3 ("CDR H3") GGYWYATSYYFDY (SEQ ID NO:5); (d) light chain CDR 1 ("CDR LI ”) RASQSISNNLN (SEQ ID NO:6); (e) light chain CDR 2 (“CDR L2”) YTSRFHS (SEQ ID NO:7); and (f) light chain CDR 3 ("CDR L3") QQEHTLPYT (SEQ ID NO:8).
  • CDR HI heavy chain CDR 1
  • CDR H2 heavy chain CDR 2
  • IIWGDGTTDYNSANKS SEQ ID ⁇ O:4
  • CDRs can be a combination of the Kabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”).
  • the CDRs comprise the Kabat CDR.
  • the CDRs are the Chothia CDR.
  • the invention provides an antibody which comprises at least one CDR that is substantially homologous to at least one CDR, at least two, at least three, at least four, at least 5 CDRs of E3 (or, in some embodiments substantially homologous to all 6 CDRs of E3, or derived from E3).
  • inventions include antibodies which have at least two, three, four, five, or six CDR(s) that are substantially homologous to at least two, three, four, five or six CDRs of E3 or derived from E3. It is understood that, for purposes of this invention, binding specificity and/or overall activity (which may be in terms of treating and/or preventing pain or inhibiting NGF-dependent survival of El 3.5 mouse trigeminal neurons) is generally retained, although the extent of activity may vary compared to E3 (may be greater or lesser).
  • the invention also provides a polypeptide (which may or may not be an antibody) which comprises an amino acid sequence of E3 (shown in Figures 1 A and IB) that has any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of a sequence of E3, wherein at least 3 of the amino acids are from a variable region of E3, with the understanding that embodiments that consist of the identical amino acid sequence to an amino acid sequence of mouse monoclonal antibody, 911, are specifically excluded.
  • E3 shown in Figures 1 A and IB
  • variable region is from a light chain of E3.
  • variable region is from a heavy chain of E3.
  • 5 (or more) contiguous amino acids are from a complementarity determining region (CDR) of E3 shown in Figures IA and IB.
  • the invention provides a polypeptide which comprises an amino acid sequence of E3 that has any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of a sequence of E3, wherein the E3 sequence comprises any one or more of: amino acid residue L29 of CDRHl, 150 of CDRH2, W101 of CDRH3, and/or A103 of CDRH3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91E of CDRL3 and/or H92 of CDRL3, with the understanding that embodiments that consist of the identical amino acid sequence to an amino acid sequence of mouse monoclonal antibody, 911, are specifically excluded.
  • a sequential amino acid numbering scheme is used to refer to amino acid residues in the variable regions (that is, the amino acid residues in each variable region are numbered in sequence).
  • the Kabat and/or Chothia numbering systems are useful when comparing two antibodies or polypeptides, such as an E3 antibody and an E3 variant (or polypeptide suspected of being an E3 variant). It is well understood in the art how to convert sequential numbering to Chothia and or Kabat numbering, if desired, for example, for use in making comparisons between E3 and another polypeptide.
  • Figure 23 depicts the E3 variable regions numbered using sequential, Chothia and Kabat numbering.
  • framework residues generally, but not always, have approximately the same number of residues.
  • the CDRs may vary in size (i.e., it is possible to have insertions and/or deletions of one or more amino acid residues).
  • an E3 antibody and a candidate E3 variant for example, in the case of a CDR region from a candidate sequence which is longer in the sequence in antibody E3 to which is is aligned
  • the candidate antibody sequence is aligned with E3 antibody heavy chain and light chain variable regions. Alignment may be done by hand, or by computer using commonly accepted computer programs.
  • Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences.
  • the light and heavy chains each typically have two cysteines, which are often found at a conserved position. It is understood tht the amino acid sequence of a candidate variant antibody may be longer (i.e. have inserted amin aci residues) or shorter (have deleted amino acid residues). Suffixes may be added to the residue number to indicate the insertion of additional residues, e.g., residue 34 abc.
  • residue 35 is simply not assigned to a residue.
  • comparison may be made between structural equivalent (e.g., same position in the antigen-antibody complex) amino acids when comparing CDRs of different lengths.
  • structural equivalent e.g., same position in the antigen-antibody complex
  • the Chothia numbering Al-Lazikani et al, supra
  • Structural equivalence may also be deduced or demonstrated using X-ray crystallography or double mutant cycle analysis (see Pons et al. (1999) Prot. Sci. 8:958-968).
  • the binding affinity of an anti-NGF antibody to NGF can be about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nM and about 0.18 to about 0.72 nM. In some embodiments, the binding affinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM. In one embodiment, the binding affinity is between about 2 pM and 22 pM.
  • the binding affinity is less than about 10 nM, about 5 nM, about 4 nnM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM. In some embodiments, the binding affinity is about 10 nM.
  • the binding affinity is less than about 10 nM. In other embodiments, the binding affinity is about 0.1 nM or about 0.07 nM. In other embodiments, the binding affinity is less than about 0.1 nM or less than about 0.07 nM.
  • the binding affinity is any of about 10 nM, about 5 nM, about 4 nnM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM to any of about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM.
  • the binding affinity is any of about 10 nM, about 5 nM, about 4 nnM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM.
  • the binding affinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM.
  • the binding affinity of the antibody to NGF can be determined using methods well known in the art. One way of determining binding affinity of antibodies to NGF is by measuring affinity of monofunctional Fab fragments of the antibody, as described in the Examples. To obtain monofunctional Fab fragments, an antibody (for example, IgG) can be cleaved with papain or expressed recombinantly.
  • the affinity of an anti-NGF Fab fragment of an antibody can be determined by surface plasmon resonance (BlAcore3000TM surface plasmon resonance (SPR) system, BIAcore, INC, Piscaway NJ), as described in the Examples.
  • This protocol is suitable for use in determining binding affinity of an antibody to NGF of any species, including human NGF, NGF of another vertebrate (in some embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF), as well as for use with other neurotrophins, such as the related neurotrophins NT3, NT4/5, and/or BDNF.
  • the antibodies or peptides of the invention may inhibit (reduce, and/or block) human NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or less.
  • the antibodies or peptides of the invention may inhibit (reduce, and/or block) human NGF- dependent survival of mouse E13.5 trigeminal neurons with an IC50 (in the presence of about 1.5 pM of NGF) of about any of 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, or less.
  • the antibodies or peptides of the invention may inhibit (reduce, and/or block) rat NGF-dependent survival of mouse El 3.5 trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF) of about any of 150 pM, 125 pM, 100 pM, 80 pM, 60 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or less.
  • the antibodies or peptides of the invention may inhibit (reduce, and/or block) rat NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 (in the presence of about 1.5 pM of NGF) of about any of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, or less.
  • Methods for measurement of the NGF-dependent survival of mouse E13 trigeminal neurons are known in the art, and described, e.g., in Example 2.
  • the invention also provides methods of making any of these antibodies or polypeptides.
  • the antibodies of this invention can be made by procedures known in the art, some of which are illustrated in the Examples.
  • the polypeptides can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis.
  • Polypeptides of the antibodies, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available.
  • a E3 antibody could be produced by an automated polypeptide synthesizer employing the solid phase method. See also, U.S. Patent Nos.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • the antibodies can be made recombinantly using procedures that are well known in the art.
  • a polynucleotide comprising a sequence encoding the variable and light chain regions of antibody E3 is cloned into a vector for expression or propagation in a host cell (e.g., CHO cells).
  • a host cell e.g., CHO cells
  • the polynucleotide sequences shown in Figures 2 and 3 are cloned into one or more vectors for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.
  • Vectors (including expression vectors) and host cells are further described herein. Methods for expressing antibodies recombinantly in plants or milk have been disclosed.
  • the invention also encompasses single chain variable region fragments
  • Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide.
  • An example of a linking peptide is (GGGGS)3 (SEQ ID NO:15), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region.
  • Linkers of other sequences have been designed and used (Bird et al. (1988)). Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports.
  • the single chain variants can be produced either recombinantly or synthetically.
  • an automated synthesizer can be used for synthetic production of scFv.
  • a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli.
  • eukaryotic such as yeast, plant, insect or mammalian cells
  • prokaryotic such as E. coli.
  • Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides.
  • the resultant scFv can be isolated using standard protein purification techniques known in the art.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., HoUiger, P., et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
  • the antibody may be a bispecific antibody, a monoclonal antibody that has binding specificities for at least two different antigens.
  • a bisecific antibody can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, the recombinant production of bispecific antibodies was based on the coexpression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, 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 (CHI), containing the site necessary for light chain binding, present in at least one of the fusions.
  • CHI first heavy chain constant region
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
  • Heteroconjugate antibodies comprising two covalently joined antibodies, are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents and techniques are well known in the art, and are described in U.S. Patent No. 4,676,980.
  • the antibody may be a humanized antibody, for example, as known in the art, and as described herein.
  • Antibodies may be modified as described in PCT Publication No. WO 2011/001100600A1
  • WO 2011/001100A1 PCT Publication No. WO 2011/001100A1
  • These antibodies comprise, in addition to a binding domain directed at the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain.
  • an effector domain having an amino acid sequence substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain.
  • the effector domain is capable of specifically binding FcRn and/or Fc ⁇ RIIb.
  • These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are preferred for use in chronic antibody therapy, to avoid inflammatory and other adverse reactions to conventional antibody therapy.
  • the invention encompasses modifications to antibody E3, including functionally equivalent antibodies which do not significantly affect their properties and variants which have enhanced or decreased activity. Modification of polypeptides is routine practice in the art and is further exemplified in the Examples. Examples of modified polypeptides include polypeptides with substitutions (including conservative substitutions) of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from a native protein in one or more substitutions, deletions, additions and/or insertions, such that the immunoreactivity of the polypeptide is not substantially diminished.
  • the ability of a variant to specifically bind antigen may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein.
  • Polypeptide variants preferably exhibit at least about 80%, more preferably at least about 90% and most preferably at least about 95% identity (determined as described herein) to the identified polypeptides.
  • Amino acid sequence variants of the antibodies may be prepared by introducing appropriate nucleotide changes into the antibody DNA, or by peptide synthesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of SEQ ID NO:l or 2 described herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis or modification is called "alanine scanning mutagenesis," and is described by Cunningham and Wells, 1989, Science, 244:1081-1085.
  • a residue or group of target residues is identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.
  • Library scanning mutagenesis as described herein, may also be used to identify locations in an antibody that are suitable for mutagenesis or modification.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.
  • Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the. products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • Non-conservative substitutions are made by exchanging a member of one of these classes for another class.
  • cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.
  • cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.
  • Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes in the variable region can alter binding affinity and/or specificity. In some embodiment, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR3 domain. In still other embodiments, the CDR domain is CDRH3 and/or CDR L3.
  • Modifications also include glycosylated and nonglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation.
  • Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).
  • the oligosaccharide side chains of the immunoglobulins affect the protein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
  • Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC).
  • CHO cells with tetracycline-regulated expression of ⁇ (l,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al., 1999, Mature Biotech. 17:176-180).
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N- linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites are conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above- described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • the glycosylation pattern of antibodies may also be altered without altering the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, variations in the glycosylation pattern of the antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol. Chem. 272:9062-9070).
  • factors that affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like.
  • Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U. S. Patent Nos. 5,047,335; 5,510,261 and 5,278,299).
  • Glycosylation, or certain types of glycosylation can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H).
  • the recombinant host cell can be genetically engineered to be defective in processing certain types of polysaccharides.
  • Modifications include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay. Modified E3 polypeptides are made using established procedures in the art and can be screened using standard assays known in the art, some of which are described below and in the Examples.
  • antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572, published November 18, 1999. These antibodies comprise, in addition to a binding domain directed at the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding the target molecule without triggering significant complement dependent lysis, or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or Fc ⁇ RIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy, to avoid inflammatory and other adverse reactions to conventional antibody therapy.
  • the invention also encompasses fusion proteins comprising one or more fragments or regions from the antibodies (such as E3) or polypeptides of this invention.
  • a fusion polypeptide is provided that comprises at least 10 contiguous amino acids of the variable light chain region shown in Figure IB and/or at least 10 amino acids of the variable heavy chain region shown in Figure 1 A.
  • the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region of E3, as shown in Figures 1 A and IB.
  • the fusion polypeptide comprises one or more CDR(s) of E3.
  • the fusion polypeptide comprises CDR H3 and/or CDR L3 of antibody E3.
  • the fusion polypeptide comprises any one or more of: amino acid residue L29 of CDRHl, 150 of CDRH2, W101 of CDRH3, and/or A103 of CDRH3; and/or amino acid residue S28 of CDRLl, N32 of CDRL1, T51 of CDRL2, 91E of CDRL3 and/or H92 of CDRL3.
  • a E3 fusion protein contains one or more E3 antibodies and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region.
  • heterologous sequences include, but are not limited to a "tag" such as a FLAG tag or a 6His tag. Tags are well known in the art.
  • a E3 fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly.
  • the E3 fusion proteins of this invention are made by preparing an expressing a polynucleotide encoding them using recombinant methods described herein, although they may also be prepared by other means known in the art, including, for example, chemical synthesis.
  • This invention also provides compositions comprising E3 antibodies or polypeptides conjugated (for example, linked) to an agent that facilitate coupling to a solid support (such as biotin or avidin).
  • a solid support such as biotin or avidin
  • Conjugation generally refers to linking these components as described herein.
  • the linking (which is generally fixing these components in proximate association at least for administration) can be achieved in any number of ways.
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • an antibody or polypeptide of this invention may be linked to a labeling agent (alternatively termed "label") such as a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • Labels are known in the art which generally provide (either directly or indirectly) a signal. Accordingly, the invention includes labeled antibodies and polypeptides.
  • the ability of the antibodies and polypeptides of this invention, such as binding NGF; reducing or inhibiting a NGF biological activity; reducing and/or blocking NGF-induced survival of El 3.5 mouse trigeminal neurons, may be tested using methods known in the art, some of which are described in the Examples.
  • the invention also provides compositions (including pharmaceutical compositions) and kits comprising antibody E3, and, as this disclosure makes clear, any or all of the antibodies and/or polypeptides described herein.
  • the invention also provides isolated polynucleotides encoding the antibodies and polypeptides of the invention (including an antibody comprising the polypeptide sequences of the light chain and heavy chain variable regions shown in Figures
  • IA and IB vectors and host cells comprising the polynucleotide.
  • the invention provides polynucleotides (or compositions, including pharmaceutical compositions), comprising polynucleotides encoding any of the following: (a) antibody E3; (b) a fragment or a region of the antibody E3; (c) a light chain of the antibody E3 as shown in Figures IB; (d) a heavy chain of the antibody E3 as shown in Figures 1 A; (e) one or more variable region(s) from a light chain and/or a heavy chain of the antibody E3; (f) one or more CDR(s) (one, two, three, four, five or six CDRs) of antibody E3 shown in Figures 1 A and IB; (g) CDR H3 from the heavy chain of antibody E3 shown in figure IA; (h) CDR L3 from the light chain of antibody E3 shown in Figure IB; (i) three CDRs from the light chain of antibody E3 shown in Figure IB; (j) three CDRs from the heavy chain of antibody E3
  • the polynucleotide comprises either or both of the polynucleotide(s) shown in Figures 2 and 3.
  • the invention is an isolated polynucleotide that encodes for an E3 light chain with a deposit number of ATCC No. PTA-4893 or ATCC No. PTA- 4894.
  • the invention is an isolated polynucleotide that encodes for an E3 heavy chain with a deposit number of ATCC No. PTA-4895.
  • the invention is an isolated polynucleotide comprising (a) a variable region encoded in the polynucleotide with a deposit number of ATCC No.
  • the invention is an isolated polynucleotide comprising (a) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA-4894; and/or (b) one or more CDR encoded in the polynucleotide with a deposit number of ATCC No. PTA- 4895.
  • the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein. Polynucleotides can be made by procedures known in the art [0203]
  • the invention provides compositions (such as a pharmaceutical compositions) comprising any of the polynucleotides of the invention.
  • the composition comprises an expression vector comprising a polynucleotide encoding the E3 antibody as described herein.
  • the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein.
  • the composition comprises either or both of the polynucleotides shown in Figures 2 and 3. Expression vectors, and administration of polynucleotide compositions are further described herein.
  • the invention provides a method of making any of the polynucleotides described herein.
  • Polynucleotides complementary to any such sequences are also encompassed by the present invention.
  • Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a portion thereof) or may comprise a variant of such a sequence.
  • Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule.
  • the effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein.
  • Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native antibody or a portion thereof.
  • Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the
  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • Variants may also, or alternatively, be substantially homologous to a native gene ; or a portion or complement thereof.
  • Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).
  • Suitable “moderately stringent conditions” include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0. 1 % SDS.
  • highly stringent conditions or “high stringency conditions” are those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sul
  • formamide for example, 50% (v
  • polynucleotides of this invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.
  • a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein.
  • Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome.
  • the polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al. (1989).
  • PCR allows reproduction of DNA sequences.
  • PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).
  • RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art, as set forth in Sambrook et al., (1989), for example.
  • Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art.
  • cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, m ⁇ l8, m ⁇ l9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invifrogen.
  • Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the invention. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462.
  • Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • the vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
  • infection e.g., where the vector is an infectious agent such as vaccinia virus.
  • the invention also provides host cells comprising any of the polynucleotides described herein.
  • Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
  • mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462.
  • Suitable non- mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis).
  • the host cells express the cDNAs at a level of about 5 fold higher, more preferably 10 fold higher, even more preferably 20 fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells.
  • Screening the host cells for a specific binding to NGF is effected by an immunoassay or FACS.
  • a cell overexpressing the antibody or protein of interest can be identified.
  • Antibody E3 which binds NGF may be used to identify or detect the presence or absence of NGF.
  • Detection generally involves contacting a biological sample with an antibody described herein that binds to NGF and the formation of a complex between NGF and an antibody (e.g., E3) which binds specifically to NGF. The formation of such a complex can be in vitro or in vivo.
  • detection includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.
  • any of a variety of known methods can be used for detection, including, but not limited to, immunoassay, using antibody that binds the polypeptide, e.g. by enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and the like; and functional assay for the encoded polypeptide, e.g. binding activity or enzymatic assay.
  • the antibody is detectably labeled.
  • Antibodies and polypeptides of the invention can be used in the detection, diagnosis and monitoring of a disease, condition, or disorder associated with altered or aberrant NGF expression (in some embodiments, increased or decreased NGF expression (relative to a normal sample), and/or inappropriate expression, such as presence of expression in tissue(s) and/or cell(s) that normally lack NGF expression, or absence of NGF expression in tissue(s) or cell(s) that normally possess NGF expression).
  • the antibodies and polypeptides of the invention are further useful for detection of NGF expression, for example, in a disease associated with altered or aberrant sensitivity or responsiveness to NGF.
  • NGF expression is detected in a sample from an individual suspected of having a disease, disorder featuring or associated with an altered or aberrant sensitivity or responsiveness to NGF expression (e.g., a cancer in which NGF promotes growth and/or metastasis).
  • the invention provides methods comprising contacting a specimen (sample) of an individual suspected of having altered or aberrant NGF expression with an antibody or polypeptide of the invention and determining whether the level of NGF differs from that of a control or comparison specimen.
  • the individual has a cardiac arrhythmia, Alzheimer's disease, and/or autonomic dysfunction.
  • the invention provides methods comprises contacting a specimen (sample) of an individual and determining level of NGF expression.
  • the individual is suspected of having a disease, disorder featuring or associated with an altered or aberrant sensitivity or responsiveness to NGF expression.
  • the individual has small cell lung cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian carcinoma, hepatocellular carcinoma, or melanoma.
  • the antibody typically will be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels. Methods of conjugating labels to an antibody are known in the art.
  • antibodies of the invention need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the antibodies of the invention.
  • the antibodies of the present invention may be employed in any known assay method, such competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).
  • the antibodies may also be used for in vivo diagnostic assays, such as in vivo imaging.
  • the antibody is labeled with a radionuclide (such as ⁇ In, 99 Tc,
  • the antibody may also be used as staining reagent in pathology, following techniques well known in the art.
  • Antibody E3 is useful for reducing and/or blocking the biological activity of
  • the invention provides a method of antagonizing human NGF biological activity using any of the polypeptides (including antibodies such as antibody E3) disclosed herein.
  • the method comprises contacting human nerve growth factor with any of the polypeptides (including antibody E3) described herein, whereby human nerve growth factor activity is antagonized, reduced, blocked, or suppressed.
  • an individual with pain (such as post-surgical pain, or rheumatoid arthritis pain) is given treatment with E3.
  • E3 or antibody For simplicity, reference will be made generally to E3 or antibody with the understanding that these methods apply to any of the E3 variant antibodies and polypeptides described herein.
  • E3 or fragments of E3 e.g., Fab, Fab' , F(ab')2, Fv,
  • E3 antibodies or various formulations of E3 thereof may be administered neat.
  • E3 or various formulations of E3 (including any composition embodiment described herein) thereof and a pharmaceutically acceptable excipient are administered, and may be in various formulations.
  • Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000). [0234] In some embodiments, these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.), although other forms of administration (e.g., oral, mucosal, via inhalation, sublingually, etc) can be also used.
  • injection e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.
  • other forms of administration e.g., oral, mucosal, via inhalation, sublingually, etc
  • E3 antibody and equivalents thereof are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • the particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
  • any of the following doses may be used: a dose of at least about 50 mg/kg body weight; at least about 10 mg/kg body weight; at least about 3 mg/kg body weight; at least about 1 mg/kg body weight; at least about 750 ⁇ g/kg body weight; at least about 500 ⁇ g/kg body weight; at least about 250 ug/kg body weight; at least about 100 ⁇ g /kg body weight; at least about 50 ⁇ g /kg body weight; at least about 10 ug /kg body weight; at least about 1 ⁇ g/kg body weight, or less, is administered.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the anti-NGF antibody, or followed by a maintenance dose of about 1 mg/kg every other week.
  • other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
  • Empirical considerations, such as the half- life, generally will contribute to determination of the dosage. The progress of this therapy is easily monitored by conventional techniques and assays.
  • Frequency of administration may be determined and adjusted over the course of therapy.
  • frequency of administration may be determined or adjusted based on the type and severity of the pain to be treated, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician.
  • an anti-NGF antagonist antibody such as E3
  • sustained continuous release formulations of E3 antibodies may be appropriate.
  • formulations and devices for achieving sustained release are known in the art.
  • dosages for E3 antibodies may be determined empirically in individuals who have been given one or more administration(s). Individuals are given incremental dosages of E3. To assess efficacy of E3 or other equivalent antibody, markers of the disease symptoms (such as pain) can be monitored. [0237] Administration of an antibody (such as E3) or polypeptide in accordance with the method in the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing pain, before, during, before and after, during and after, or before, during, and after developing pain. Administration can be before, during and/or after wound, incision, trauma, surgery, and any other event likely to give rise to post-surgical pain.
  • compositions include suitable delivery forms known in the art including, but not limited to, carriers such as liposomes. See, for example, Mahato et al. (1997) Pharm. Res. 14:853-859.
  • Liposomal preparations include, but are not limited to, cytofectins, multilamellar vesicles and unilamellar vesicles.
  • more than one antibody or polypeptide may be present.
  • the antibodies can be monoclonal or polyclonal.
  • Such compositions may contain at least one, at least two, at least three, at least four, at least five different antibodies.
  • a mixture of antibodies, as they are often denoted in the art, may be particularly useful in treating a broader range of population of individuals.
  • a polynucleotide encoding any of the antibodies or polypeptides of the invention may also be used for delivery and expression of any of the antibodies or polypeptides of the invention (such as antibody E3) in a desired cell.
  • an expression vector can be used to direct expression of an E3 antibody or polypeptide.
  • the expression vector can be administered by any means known in the art, such as intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, dermally, sublingually, or by inhalation.
  • administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • local or systemic administration including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471.
  • Targeted delivery of therapeutic compositions comprising a polynucleotide encoding any of the antibodies or polypeptides of the invention (such as antibody E3) can also be used.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA can also be used during a gene therapy protocol.
  • the therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
  • Niral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication ⁇ os. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Patent ⁇ os. 5, 219,740; 4,777,127; GB Patent No. 2,200,651; and EP Patent No.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC NR-1247), Ross River virus (ATCC NR-373; ATCC NR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno-associated virus
  • ⁇ on- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed D ⁇ A linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked D ⁇ A(see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes.
  • polycationic condensed D ⁇ A linked or unlinked to killed adenovirus alone see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147
  • ligand-linked D ⁇ A see, e.g., Wu, J. Bio
  • Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent NO. 0 524 968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
  • reference to anti-NGF antagonist antibodies also include compositions comprising one or more of these agents. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art. The present invention can be used alone or in combination with other conventional methods of treatment.
  • the invention provides methods for treating and/or preventing rheumatoid arthritis pain in individuals including mammals, both human and non-human. Accordingly, in one aspect, the invention provides methods of treating rheumatoid arthritis pain in an individual comprising administering an effective amount of an anti-NGF antagonist antibody.
  • Anti-NGF antagonist antibodies are known in the art and described herein.
  • the invention provides methods for reducing incidence of, ameliorating, suppressing, palliating, and/or delaying the onset, the development or the progression of rheumatoid arthritis pain in an individual.
  • the anti-NGF antagonist antibody is administered prior to development of pain or a pain episode in an individual having rheumatoid arthritis.
  • the invention provides methods for treating inflammatory cachexia (weight loss) associated with rheumatoid arthritis in an individual comprising administering an effective amount of an anti-NGF antagonist antibody (Roubenoff et al., Arthritis Rheum. 40(3): 534-9 (1997); Roubenoff et al, J. Clin. Invest. 93(6):2379-86 (1994)).
  • Diagnosis or assessment of rheumatoid arthritis pain is well-established in the art. Assessment may be performed based on measures known in the art, such as patient characterization of pain using various pain scales. See, e.g., Katz et al, Surg Clin North Am. (1999) 79 (2):231-52; Caraceni et al. JPain Symptom Manage (2002) 23(3):239-55.
  • Anti-NGF antagonist antibody may be administered to an individual via any suitable route. Examples of different administration route are described herein.
  • Pain relief may be characterized by time course of relief. Accordingly, in some embodiments, pain relief is observed within about 24 hours after administration of anti-NGF antagonist antibody. In other embodiments, pain relief is observed within about 36, 48, 60, 72 hours or 4 days after administration of anti-NGF antagonist antibody. In still other embodiments, pain relief is observed before observing an indication of improvement of the inflammatory condition associated with rheumatoid arthritis. In some embodiments, frequency and/or intensity of pain is diminished, and/or quality of life of those suffering the the disease is increased.
  • the invention provides methods for treating and/or preventing osteoarthritis pain in individuals including mammals, both human and non- human. Accordingly, in one aspect, the invention provides methods of treating osteoarthritis pain in an individual comprising administering an effective amount of an anti- NGF antagonist antibody.
  • Anti-NGF antagonist antibodies are known in the art and described herein.
  • the invention provides methods for reducing incidence of, ameliorating, suppressing, palliating, and/or delaying the onset, the development or the progression of osteoarthritis pain in an individual.
  • the anti- NGF antagonist antibody is administered prior to development of pain or a pain episode in an individual having osteoarthritis.
  • WOMAC Ambulation Pain Scale including pain, stiffness, and physical function
  • NAS 100 mm Visual Analogue Scale
  • Anti- ⁇ GF antagonist antibody may be administered to an individual via any suitable route. Examples of different administration route are described herein.
  • Pain relief may be characterized by time course of relief. Accordingly, in some embodiments, pain relief is observed within about 24 hours after administration of anti- ⁇ GF antagonist antibody. In other embodiments, pain relief is observed within about
  • frequency and/or intensity of pain is diminished, and/or quality of life of those suffering the the disease is increased.
  • Anti- ⁇ GF antagonist antibody refers to any antibody molecule that blocks, suppresses or reduces (including significantly) ⁇ GF biological activity, including downstream pathways mediated by ⁇ GF signaling, such as receptor binding and/or elicitation of a cellular response to ⁇ GF.
  • An anti- ⁇ GF antagonist antibody should exhibit any one or more of the following characteristics: (a) bind to ⁇ GF and inhibit ⁇ GF biological activity or downstream pathways mediated by ⁇ GF signaling function; (b) prevent, ameliorate, or treat any aspect of rheumatoid arthritis pain or osteoarthritis pain; (c) block or decrease ⁇ GF receptor activation (including TrkA receptor dimerization and/or autophosphorylation); (d) increase clearance of ⁇ GF; (e) inhibit (reduce) ⁇ GF synthesis, production or release.
  • Anti- ⁇ GF antagonist antibodies are known in the art, see, e.g., PCT Publication ⁇ os. WO 01/78698, WO 01/64247, U.S. Patent ⁇ os.
  • the antibody reacts with ⁇ GF in a manner that inhibits ⁇ GF and/or downstream pathways mediated by the ⁇ GF signaling function.
  • the anti-NGF antagonist antibody recognizes human NGF.
  • the anti-NGF antagonist antibody specifically binds human NGF.
  • the anti-NGF antagonist antibody does not significantly bind to related neurotrophins, such as NT-3, NT4/5, and/or BDNF.
  • the anti- NGF antibody is capable of binding NGF and effectively inhibiting the binding of NGF to its TrkA and/or p75 receptor in vivo and/or effectively inhibiting NGF from activating its TrkA and/or p75 receptor.
  • the anti-NGF antagonist antibody is a monoclonal antibody.
  • the anti-NGF antibody is humanized (such as antibody E3 described herein). In some embodiments, the anti-NGF antibody is human.
  • the antibody is a human antibody which recognizes one or more epitopes on human NGF.
  • the antibody is a mouse or rat antibody which recognizes one or more epitopes on human NGF.
  • the antibody recognizes one or more epitopes on an NGF selected from the group consisting of: primate, canine, feline, equine, and bovine.
  • the anti-NGF antagonist antibody binds essentially the same NGF epitope 6 as an antibody selected from any one or more of the following: MAb 911, MAb 912 and MAb 938 (See Hongo, et al., Hybridoma 19:215-227 (2000)).
  • the antibody binds the same epitope as Mab 911 ;
  • the antibody comprises a constant region that is immunologically inert (e.g., does not trigger complement mediated lysis or antibody dependent cell mediated cytotoxicity (ADCC)). ADCC activity can be assessed using methods disclosed in U.S. Patent NO. 5, 500, 362.
  • the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.
  • the anti-NGF antagonist antibody is a humanized mouse anti-NGF monoclonal antibody termed antibody "E3", any of the E3 related antibodies described herein, or any fragments thereof, which are NGF antagonists.
  • the antibodies useful in the present invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • the antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).
  • the binding affinity of an anti-NGF antagonist antibody to NGF can be about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nM and about 0.18 to about 0.72 nM. In one embodiment, the binding affinity is between about 2 pM and 22 pM. In some embodiment, the binding affinity is about 10 nM. In other embodiments, the binding affinity is less than about 10 nM. In other embodiments, the binding affinity is about 0.1 nM or about 0.07 nM. In other embodiments, the binding affinity is less than about 0.1 nM or less than about 0.07 nM.
  • the binding affinity is any of about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM to any of about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM. In some embodiments, the binding affinity is any of about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM, or less than about 50 pM.
  • the binding affinity is less than any of about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM. In still other embodiments, the binding affinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM.
  • One way of determining binding affinity of antibodies to NGF is by measuring binding affinity of monofunctional Fab fragments of the antibody. To obtain monofunctional Fab fragments, an antibody (for example, IgG) can be cleaved with papain or expressed recombinantly.
  • CM5 chips can be activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions.
  • EDC N-ethyl-N'- (3-dimethylaminopropyl)-carbodiinide hydrochloride
  • NHS N-hydroxysuccinimide
  • Equilibrium dissociation constant (K D ) values are calculated as koff/k o n- This protocol is suitable for use in determining binding affinity of an antibody to any NGF, including human NGF, NGF of another vertebrate (in some embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF), as well as for use with other neurotrophins, such as the related neurotrophins NT3, NT4/5, and/or BDNF.
  • the antibody binds human NGF, and does not significantly bind an NGF from another vertebrate species (in some embodiment, mammalian). In some embodiments, the antibody binds human NGF as well as one or more NGF from another vertebrate species (in some embodiments, mammalian). In still other embodiments, the antibody binds NGF and does not significantly cross-react with other neurotrophins (such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In some embodiments, the antibody binds NGF as well as at least one other neurotrophin.
  • other neurotrophins such as the related neurotrophins, NT3, NT4/5, and/or BDNF.
  • the antibody binds to a mammalian species of NGF, such as horse or dog, but does not significantly bind to NGF from anther mammalian species.
  • the epitope(s) can be continuous or discontinuous.
  • the antibody binds essentially the same hNGF epitopes as an antibody selected from the group consisting of MAb 911, MAb 912, and MAb 938 as described in Hongo et al., Hybridoma, 19:215-227 (2000).
  • the antibody binds essentially the same hNGF epitope as MAb 911.
  • the antibody binds essentially the same epitope as MAb 909. Hongo et al., supra.
  • the epitope may comprise one or more of: residues K32, K34 and E35 within variable region 1 (amino acids 23-35) of hNGF; residues F79 and T81 within variable region 4 (amino acids 81-88) of hNGF; residues H84 and K88 within variable region 4; residue R103 between variable region 5 (amino acids 94-98) of hNGF and the C-terminus (amino acids 111-118) of hNGF; residue Ell within pre- variable region 1 (amino acids 10-23) of hNGF; Y52 between variable region 2 (amino acids 40-49) of hNGF and variable region 3 (amino acids 59-66) of hNGF; residues LI 12 and SI 13 within the C-terminus of hNGF; residues R59 and R69 within variable region 3 of hNGF; or residues VI 8, N20, and G23 within pre-variable region 1 of h ⁇ GF.
  • an epitope can comprise one or more of the variable region 1, variable region 3, variable region 4, variable region 5, the ⁇ -terminus region, and /or the C-terminus of h ⁇ GF.
  • the antibody significantly reduces the solvent accessibility of residue R103 of h ⁇ GF. It is understood that although the epitopes described above relate to human ⁇ GF, one of ordinary skill can align the structures of human ⁇ GF with the ⁇ GF of other species and identify likely counterparts to these epitopes.
  • antibodies that can inhibit ⁇ GF may be made by using immunogens that express full length or partial sequence of ⁇ GF.
  • an immunogen comprising a cell that overexpresses ⁇ GF may be used.
  • Another example of an immunogen that can be used is ⁇ GF protein that contains full-length ⁇ GF or a portion of the ⁇ GF protein.
  • the anti- ⁇ GF antagonist antibodies may be made by any method known in the art.
  • the route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein.
  • General techniques for production of human and mouse antibodies are known in the art and are described herein.
  • any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human, hybridoma cell lines.
  • the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.
  • Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif, USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art.
  • a fusogen such as polyethylene glycol
  • the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin- thymidine (HAT) medium, to eliminate unhybridized parent cells.
  • a selective growth medium such as hypoxanthine-aminopterin- thymidine (HAT) medium
  • HAT hypoxanthine-aminopterin- thymidine
  • Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies.
  • EBV immortalized B cells may be used to produce the anti-NGF monoclonal antibodies of the subject invention.
  • hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
  • immunoassay procedures e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay.
  • Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies specific for NGF, or a portion thereof.
  • Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
  • the monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
  • Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean try
  • the anti-NGF antagonist antibody (monoclonal or polyclonal) of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence may be used for genetic manipulation to "humanize" the antibody or to improve the affinity, or other characteristics of the antibody.
  • the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans.
  • a number of "humanized" antibody molecules comprising an antigen- binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains.
  • CDRs complementarity determining regions
  • rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain See, for example, Riechmann et al. Nature 332:323-327 (1988), Verhoeyen et al. Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525 (1986).
  • Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, for example, European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize unwanted immunological response toward rodent anti-human antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
  • the antibody constant region can be engineered such that it is immunologically inert (e.g., does not trigger complement lysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK Patent Application No. 9809951.8.
  • Other methods of humanizing antibodies that may also be utilized are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991) and in U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01/27160.
  • Fully human antibodies may be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse TM from Abgenix, Inc. (Fremont, CA) and HuMAb-Mouse® and TC MouseTM from Medarex, Inc. (Princeton, NJ).
  • antibodies may be made recombinantly and expressed using any method known in the art.
  • antibodies may be made recombinantly by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Anna Rev. Immunol. 12:433-455 (1994).
  • the phage display technology McCafferty et al, Nature 348:552- 553 (1990)
  • V immunoglobulin variable
  • antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
  • a filamentous bacteriophage such as Ml 3 or fd
  • the filamentous particle contains a single-stranded DNA copy of the phage genome
  • selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B cell.
  • Phage display can be performed in a variety of formats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).
  • V-gene segments can be used for phage display.
  • Clackson et al Nature 352:624-628 (1991) isolated a diverse array of anti- oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Mark et al, J. Mol. Biol. 222:581-597 (1991), or Griffith et al, EMBO J. 12:725-734 (1993).
  • antibody genes accumulate mutations at a high rate (somatic hypermutation).
  • Antibodies may be made recombinantly by first isolating the antibodies and antibody producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method which may be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for expressing antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D.
  • Immunoassays and flow cytometry sorting techniques such as fluorescence activated cell sorting (FACS) can also be employed to isolate antibodies that are specific for NGF.
  • FACS fluorescence activated cell sorting
  • the antibodies can be bound to many different carriers.
  • Ca ⁇ iers can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
  • the carrier comprises a moiety that targets the myocardium.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors (such as expression vectors disclosed in PCT Publication No. WO 87/04462), which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • expression vectors such as expression vectors disclosed in PCT Publication No. WO 87/04462
  • host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In that manner, "chimeric" or “hybrid” antibodies are prepared that have the binding specificity of an anti-NGF monoclonal antibody herein. [0282] Anti-NGF antagonist antibodies may be characterized using methods well known in the art.
  • one method is to identify the epitope to which it binds, or "epitope mapping.”
  • epitope mapping There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.
  • epitope mapping can be used to determine the sequence to which an anti-NGF antagonist antibody binds.
  • Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands).
  • the epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch.
  • Peptides of varying lengths e.g., at least 4-6 amino acids long
  • the epitope to which the anti-NGF antagonist antibody binds can be determined in a systematic screening by using overlapping peptides derived from the NGF sequence and determining binding by the anti- NGF antagonist antibody.
  • the open reading frame encoding NGF is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of NGF with the antibody to be tested is determined.
  • the gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled NGF fragments is then determined by immunoprecipitation and gel electrophoresis.
  • Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays.
  • mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding.
  • domain swapping experiments can be performed using a mutant NGF in which various fragments of the NGF polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein (such as another member of the neurotrophin protein family). By assessing binding of the antibody to the mutant NGF, the importance of the particular NGF fragment to antibody binding can be assessed.
  • Yet another method which can be used to characterize an anti-NGF antagonist antibody is to use competition assays with other antibodies known to bind to the same antigen, i.e., various fragments on NGF, to determine if the anti-NGF antagonist antibody binds to the same epitope as other antibodies.
  • Competition assays are well known to those of skill in the art.
  • Example of antibodies that can be used in the competition assays for the present invention include MAb 911, 912, 938, as described in Hongo, et al., Hybridoma 19:215-227 (2000).
  • An expression vector can be used to direct expression of an anti-NGF antagonist antibody.
  • One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471.
  • Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • the expression vector is administered directly to the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle, or pericardium.
  • Targeted delivery of therapeutic compositions containing an expression vector, or subgenomic polynucleotides can also be used.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA can also be used during a gene therapy protocol.
  • the therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Patent Nos. 5, 219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR- 1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno-associated virus
  • Non- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos.
  • WO 95/07994 WO 96/17072; WO 95/30763; and WO 97/423378
  • nucleic charge neutralization or fusion with cell membranes can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci.
  • Anti-NGF antagonist antibodies can be identified or characterized using methods known in the art, whereby reduction, amelioration, or neutralization of an NGF biological activity is detected and/or measured.
  • a kinase receptor activation (KIRA) assay described in U.S. Patent Nos. 5,766,863 and 5,891,650 can be used to identify anti-NGF agents.
  • This ELISA-type assay is suitable for qualitative or quantitative measurement of kinase activation by measuring the autophosphorylation of the kinase domain of a receptor protein tyrosine kinase (hereinafter "rPTK”), e.g.
  • rPTK receptor protein tyrosine kinase
  • TrkA receptor as well as for identification and characterization of potential antagonists of a selected rPTK, e.g., TrkA.
  • the first stage of the assay involves phosphorylation of the kinase domain of a kinase receptor, for example, a TrkA receptor, wherein the receptor is present in the cell membrane of an eukaryotic cell.
  • the receptor may be an endogenous receptor or nucleic acid encoding the receptor, or a receptor construct, may be transformed into the cell.
  • a first solid phase e.g., a well of a first assay plate
  • a substantially homogeneous population of such cells usually a mammalian cell line
  • a "receptor construct" it usually comprises a fusion of a kinase receptor and a flag polypeptide.
  • the flag polypeptide is recognized by the capture agent, often a capture antibody, in the ELISA part of the assay.
  • An analyte such as a candidate anti-NGF antagonist antibody is then added together with NGF to the wells having the adherent cells, such that the tyrosine kinase receptor (e.g. TrkA receptor) is exposed to (or contacted with) NGF and the analyte.
  • TrkA receptor tyrosine kinase receptor
  • the adhering cells are solubilized using a lysis buffer (which has a solubilizing detergent therein) and gentle agitation, thereby releasing cell lysate which can be subjected to the ELISA part of the assay directly, without the need for concentration or clarification of the cell lysate.
  • a lysis buffer which has a solubilizing detergent therein
  • the cell lysate thus prepared is then ready to be subjected to the ELISA stage of the assay.
  • a second solid phase usually a well of an ELISA microtiter plate
  • a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide.
  • Coating of the second solid phase is carried out so that the capture agent adheres to the second solid phase.
  • the capture agent is generally a monoclonal antibody, but, as is described in the examples herein, polyclonal antibodies may also be used.
  • the cell lysate obtained is then exposed to, or contacted with, the adhering capture agent so that the receptor or receptor construct adheres to (or is captured in) the second solid phase.
  • a washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct.
  • the adhering or captured receptor or receptor construct is then exposed to, or contacted with, an anti- phosphotyrosine antibody which identifies phosphorylated tyrosine residues in the tyrosine kinase receptor.
  • the anti-phosphotyrosine antibody is conjugated (directly or indirectly) to an enzyme which catalyses a color change of a non-radioactive color reagent.
  • phosphorylation of the receptor can be measured by a subsequent color change of the reagent.
  • the enzyme can be bound to the anti- phosphotyrosine antibody directly, or a conjugating molecule (e.g., biotin) can be conjugated to the anti-phosphotyrosine antibody and the enzyme can be subsequently bound to the anti-phosphotyrosine antibody via the conjugating molecule.
  • binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured, e.g., by a color change in the color reagent.
  • the anti-NGF antagonist antibody can also be identified by incubating a candidate agent with NGF and monitoring any one or more of the following characteristics: (a) binding to NGF and inhibiting NGF biological activity or downstream pathways mediated by NGF signaling function; (b) inhibiting, blocking or decreasing NGF receptor activation (including TrkA dimerization and/or autophosphorylation); (c) increasing clearance of NGF; (d) treating or preventing any aspect of rheumatoid arthritis pain or osteoarthritis pain; (e) inhibiting (reducing) NGF synthesis, production or release.
  • an anti-NGF antagonist antibody is identified by incubating an candidate agent with NGF and monitoring binding and/or attendant reduction or neutralization of a biological activity of NGF.
  • the binding assay may be performed with purified NGF polypeptide(s), or with cells naturally expressing, or transfected to express, NGF polypeptide(s).
  • the binding assay is a competitive binding assay, where the ability of a candidate antibody to compete with a known anti-NGF antagonist for NGF binding is evaluated.
  • the assay may be performed in various formats, including the ELISA format.
  • an anti-NGF antagonist antibody is identified by incubating a candidate agent with NGF and monitoring binding and attendant inhibition of trkA receptor dimerization and/or autophosphorylation.
  • NGF promotes a number of morphologically recognizable changes in responsive cells. These include, but are not limited to, promoting the differentiation of PC 12 cells and enhancing the growth of neurites from these cells (Greene et al., Proc Natl Acad Sci U S A. 73(7):2424-8, 1976), promoting neurite outgrowth from explants of responsive sensory and sympathetic ganglia (Levi-Montalcini, R. and Angeletti, P. Nerve growth factor. Physiol. Rev.
  • the assay for inhibition of NGF biological activity entail culturing NGF responsive cells with NGF plus an analyte, such as a candidate anti-NGF antagonist antibody. After an appropriate time the cell response will be assayed (cell differentiation, neurite outgrowth or cell survival).
  • the ability of a candidate anti-NGF antagonist antibody to block or neutralize a biological activity of NGF can also be assessed by monitoring the ability of the candidate agent to inhibit NGF mediated survival in the embryonic rat dorsal .root ganglia survival bioassay as described in Hongo et al, Hybridoma 19:215-227 (2000).
  • the anti-NGF antagonist antibody can be administered to an individual (for rheumatoid arthritis and osteoarthritis) via any suitable route. It should be apparent to a person skilled in the art that the examples described herein are not intended to be limiting but to be illustrative of the techniques available.
  • the anti-NGF antagonist antibody is administered to a individual in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, mtraperitoneal, intracerebrospinal, subcutaneous, intra- articular, sublingually, intrasynovial, via insufflation, intrathecal, oral, inhalation or topical routes.
  • Administration can be systemic, e.g., intravenous administration, or localized.
  • nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers are useful for administration.
  • Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
  • anti- NGF antagonist antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
  • an anti-NGF antagonist antibody is administered via site-specific or targeted local delivery techniques.
  • site-specific or targeted local delivery techniques include various implantable depot sources of the anti-NGF antagonist antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Patent No. 5,981,568. [0295] Various formulations of an anti-NGF antagonist antibody may be used for administration. In some embodiments, the anti-NGF antagonist antibody may be administered neat. In some embodiments, anti-NGF antagonist antibody and a pharmaceutically acceptable excipient may be in various formulations.
  • excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
  • Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
  • these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • the particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
  • an anti-NGF antibody can be administered using any suitable method, including by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Anti-NGF antibodies can also be administered via inhalation, as described herein. Generally, for administration of anti-NGF antibodies, an initial candidate dosage can be about 2 mg/kg. For the purpose of the present invention, a typical daily dosage might range from about any of 1 ⁇ g/kg to 3 ⁇ g/kg to 30 ⁇ g/kg to 300 ⁇ g/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • an anti-NGF antibody may be administered at about 1 ⁇ g/kg, about 10 ⁇ g/kg, about 20 ⁇ g/kg, about 50 ⁇ g/kg, about 100 ⁇ g/kg, about 200 ⁇ g/kg, about 500 ⁇ g/kg, about 1 mg/kg, or about 2 mg/kg.
  • the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to reduce pain.
  • An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the anti-NGF antibody, or followed by a maintenance dose of about 1 mg/kg every other week.
  • dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, in some embodiments, dosing from one-four times a week is contemplated. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the dosing regimen (including the NGF antagonist(s) used) can vary over time..
  • NGF antagonist antibody will depend on the anti-NGF antagonist antibody (or compositions thereof) employed, the type and severity of the pain to be treated, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. Typically the clinician will administer an anti-NGF antagonist antibody, until a dosage is reached that achieves the desired result. Dose and/or frequency can vary over course of treatment.
  • Empirical considerations such as the half-life, generally will contribute to the determination of the dosage.
  • antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of pain.
  • sustained continuous release formulations of anti-NGF antagonist antibodies may be appropriate.
  • formulations and devices for achieving sustained release are known in the art.
  • dosages for an anti-NGF antagonist antibody may be determined empirically in individuals who have been given one or more administration(s) of an anti-NGF antagonist antibody. Individuals are given incremental dosages of an anti- NGF antagonist antibody. To assess efficacy of an anti-NGF antagonist antibody, an indicator of pain can be followed.
  • Administration of an anti-NGF antagonist antibody in accordance with the method in the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of an anti-lSfGF antagonist antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing pain; before; during; before and after; during and after; before and during; or before, during, and after developing pain.
  • more than one anti-NGF antagonist antibody may be present. At least one, at least two, at least three, at least four, at least five different, or more anti-NGF antagonist antibody can be present. Generally, those anti-NGF antagonist antibodies have complementary activities that do not adversely affect each other.
  • Therapeutic formulations of the anti-NGF antagonist antibody used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may comprise buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
  • Liposomes containing the anti-NGF antagonist antibody are prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or 'poly(v nylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and 7 ethyl-L- glutamate copolymers of L-glutamic acid and 7 ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly- D-(-)-3-hydroxybutyric acid.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic anti-NGF antagonist antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions according to the present invention may be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
  • the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g. TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g. SpanTM 20, 40, 60, 80 or 85).
  • Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
  • Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. soybean oil, saffiower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • an oil e.g. soybean oil, saffiower oil, cottonseed oil, sesame oil, corn oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion can comprise fat droplets between 0.1 and 1.0 . ⁇ m, particularly 0.1 and 0.5 . ⁇ m, and have a pH in the range of 5.5 to 8.0.
  • the emulsion compositions can be those prepared by mixing a nerve growth factor antibody with IntralipidTM.or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine.
  • Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • Treatment efficacy can be assessed by methods well-known in the art.
  • kits comprising antibodies or polypeptides for use in detection and/or therapy. Accordingly, in some embodiments, the kits comprise an antibody E3. In some embodiments, the kit comprises any antibody or polypeptide described herein.
  • kits may be used for any of the methods described herein, including, for example, to treat an individual with pain (including post-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain).
  • the kits of this invention are in suitable packaging, and may optionally provide additional components such as, buffers and instructions for use of the antibody in any of the methods described herein.
  • the kits include instructions for treating pain.
  • the kit comprises an anti-NGF antagonist antibody described herein and instructions for treating and/or preventing rheumatoid arthritis pain in an individual.
  • the kit comprises an anti-NGF antagonist antibody described herein and instructions for treating and/or preventing osteoarthritis pain in an individual.
  • the anti-NGF antagonist antibody is antibody E3.
  • kits comprising a polynucleotide encoding an E3 polypeptide as described herein.
  • the kits further comprise instructions for use of the polynucleotide in any of the methods described herein.
  • library scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using art recognized methods. This generates small libraries of clones (in some embodiments, one for every amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at every position).
  • the library also includes a clone comprising the native (unsubstituted) amino acid.
  • a small number of clones, e.g., about 20-80 clones (depending on the complexity of the library), from each library are screened for binding affinity to the target polypeptide, and candidates with increased, the same, decreased or no binding are identified.
  • Methods for determining binding affinity are well-known in the art.
  • binding affinity is determined using BIAcore surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater.
  • BIAcore is particularly useful when the starting antibody already binds with a relatively high affinity, for example a K D of about 10 nM or lower. Screening using BIAcore surface plasmon resonance is described in the Examples, herein.
  • binding affinity is determined using Kinexa
  • binding affinity is screened using a suitable bioassay.
  • every amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 natural amino acids using art recognized mutagenesis methods (some of which are described herein). This generates small libraries of clones (in some embodiments, one for every amino acid position that is analyzed), each with a complexity of 20 members (if all 20 amino acids are substituted at every position).
  • the library to be screened comprises substitutions in two or more positions, which may be in the same CDR or in two or more CDRs.
  • the library comprises substitutions in two or more positions in one CDR.
  • the library comprises substitution in two or more positions in two or more CDRs.
  • the library comprises substitution in 3, 4, 5, or more positions, said positions found in two, three, four, five or six CDRs.
  • the substitution is prepared using low redundancy codons. See, e.g., Table 2 ofBalint et al. , (1993) Gene 137(1):109-18).
  • the CDR is CDRH3 and/or CDRL3. In other embodiments, the CDR is one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. In some embodiments, the CDR is a Kabat CDR, a Chothia CDR, or an extended CDR.
  • Candidates with improved binding may be sequenced, thereby identifying a
  • CDR substitution mutant which results in improved affinity also termed an "improved” substitution.
  • use of this method permitted identification of a single substitution which improved binding, even when an estimated 18 other substitutions at the same amino acid position resulted in no binding (i.e., loss of antibody function).
  • Candidates that bind may also be sequenced, thereby identifying a CDR substitution which retains binding.
  • multiple rounds of screening are conducted.
  • candidates each comprising an amino acid substitution at one or more position of one or more CDR
  • candidate binding are also useful for the design of a second library containing at least the original and substituted amino acid at each improved CDR position (i.e., amino acid position in the CDR at which a substitution mutant showed improved binding).
  • Preparation, and screening or selection of this library is discussed further below.
  • Library scanning mutagenesis also provides a means for characterizing a
  • the same binding, decreased binding or no binding also provide information relating to the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a position of the CDR retains binding when changed to all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a position of CDR retains binding in only a small percentage of substitutions, that position is identified as a position that is important to CDR function.
  • the library scanning mutagenesis methods generate information regarding positions in the CDRs that can be changed to many different amino acid (including all 20 amino acids), and positions in the CDRs which cannot be changed or which can only be changed to a few amino acids. This aspect is discussed and exemplified in Example 1.
  • candidates with improved affinity are combined in a second library, which includes the improved amino acid, the original amino acid at that position, and may further include additional substitutions at that position, depending on the complexity of the library that is desired, or permitted using the desired screening or selection method.
  • adjacent amino acid position can be randomized to at least two or more amino acids. Randomization of adjacent amino acids may permit additional conformational flexibility in the mutant CDR, which may in turn, permit or facilitate the introduction of a larger number of improving mutations.
  • the library also comprises substitution at positions that did not show improved affinity in the first round of screening.
  • the second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including screening using BIAcore surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.
  • the methods are useful for pre-screening CDR amino acid positions in order to identify amino acid substitutions that improve binding or retain binding. Pre-identifying of important residues, substitution that improve binding and/or substitutions that retain antibody function permits efficient design and screening of an affinity maturation library.
  • the present method is also useful for characterizing a CDR, and provides comprehensive information regarding the importance of each amino acid position in a CDR for binding to antigen.
  • the present method may also be used to identify substitutions that improve binding.
  • the methods of the invention permits introduction and characterization of the effect of the substitution of, for example, all 20 amino acids at a given position.
  • This analysis provides information as to how many substitutions at a given position are tolerated (i.e., retain antibody binding), which in turn, provides information relating to the importance of each amino acid for antibody function. Further, substitutions that result in improved binding can be identified, even under circumstances in which many or most of the substitutions at a given position yield non-functional (non-binding) antibodies.
  • alanine-scanning mutagenesis which is commonly used to identify important CDR positions, provides information relating to whether the substitution of alanine permits or prevents binding.
  • positions at which an alanine substitution prevents binding are removed from the affinity maturation library. In many cases, however, alanine may be a poor substitute at the CDR position.
  • the present methods also permit identification and characterization of the effect of single CDR mutations.
  • methods such as phage display introduce and select many mutations simultaneously, and thus potentially increase the risk that positive mutations will be masked by the presence of a detrimental mutation present in a particular . clone.
  • the present methods are also useful for improving affinity while retaining the binding specificity of the original (starting) antibody, insofar as the present methods permit identification of small numbers of mutations (e.g., 1, 2, 3, 4, or 5 mutations in a single CDR) that result in improved binding affinity.
  • methods such as phage display typically improve binding affinity using multiple mutations at once, which may result in shifting specificity of the antibody and/or increasing undesirable cross-reactivity.
  • Small scale Fab preparation [0337] Small scale expression in 96 wells plates was optimized for screening Fab libraries. Starting from E. coli transformed with a Fab library, colonies were picked to inoculate both a master plate (agar LB + Ampicillin (50 ⁇ g/ml) + 2% Glucose) and a working plate (2 ml/well, 96 well/plate containing 1.5 mL of LB + Ampicillin (50 ⁇ g/ml) + 2% Glucose). Both plates were grown at 30°C for 8-12 hours.
  • a master plate agar LB + Ampicillin (50 ⁇ g/ml) + 2% Glucose
  • the master plate - was stored at 4°C and the cells from the working plate were pelleted at 5000 rpm and resuspended with 1 mL of LB+ Ampicillin (50 ⁇ g/ml)+ 1 mM IPTG to induce expression of Fabs.
  • Cells were harvested by centrifugation after 5 h expression time at 30°C, then resuspended in 500 ⁇ L of buffer HBS- ⁇ P (100 mM H ⁇ P ⁇ S buffer pH 7.4, 150 mM NaCI, 0.005% P20, 3 mM ⁇ DTA).
  • Lysis of HBS- ⁇ P resuspended cells was attained by one cycle of freezing (-80°C) then thawing at 37°C Cell lysates were centrifuged at 5000 rpm for 30 min to separate cell debris from supernatants containing Fabs. The supernatants were then injected into the BIAcore plasmon resonance apparatus to obtain affinity information for each Fab. Clones expressing Fabs were rescued from the master plate to sequence the DNA and for large scale Fab production and detailed characterization as described below.
  • Fabs were expressed and purified from large cultures. ⁇ rlenmeyer flasks containing 200 mL of LB+Ampicillin (50 ⁇ g/ml) + 2% Glucose were inoculated with 5 mL of over night culture from a selected Fab- expressing E. coli clone. Clones were incubated at 30°C until an ODs 5 o nm of 1.0 was attained and then induced by replacing the media for 200 ml, of LB+Ampicillin (50 ⁇ g/ml) + 1 mM IPTG.
  • Full antibody preparation [0339] For expression of full antibodies, heavy and light chain variable regions were cloned in 2 mammalian expression vectors ( ⁇ b.911.E3 or Eb.pur.911.3E for light chain and Db.911.3E for heavy chain; described herein) and transfected using lipofectemine into HEK 293 cells for transient expression. Antibodies were purified using protein A using standard methods.
  • Biacore Assay Affinities of anti- ⁇ GF Fabs and monoclonal antibodies were determined using the BlAcore3000TM surface plasmon resonance (SPR) system (BIAcore, I ⁇ C, Piscaway ⁇ J). CM5 chips were activated with ⁇ -ethyl- ⁇ '-(3-dimethylaminopropyl)- carbodiinide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Human NGF was diluted into 10 mM sodium acetate pH 4.0 and injected over the activated chip at a concentration of 0.005 mg/mL.
  • SPR surface plasmon resonance
  • a screening BIAcore assay was optimized to determine the affinity of Fab clones from libraries. Supernatants of small culture lysates were injected at 50 ⁇ l/min for 2 min. Dissociation times of 10 to 15 minutes were used for determination of a single exponential dissociation rate (k 0 ff) using BIAevaluation software. Samples that showed k off rates in the same range as the template used to create the library
  • Clones showing improved (slower) k 0f r values were expressed at large scale and full kinetic parameters, k on and k off , were determined on purified protein.
  • the assay was capable of detecting differences in affinity that were approximately 2-fold or larger.
  • Hybridoma 19(3):215-227 was selected for humanization and affinity maturation.
  • Mab 911 binds human and rat NGF with high affinity and exhibits no significant cross-reactivity with the neurotrophins NT3, NT4/5 or BDNF. See Hongo, id.
  • the affinity of the papain- cleaved Fab fragment of mouse Mab 911 was determined using BIAcore analysis as described above.
  • the papain-cleaved Fab fragment of mouse Mab 911 bound human NGF with a K D of approximately 10 nM.
  • hVH and hVL humanized heavy and light variable domains were termed hVH and hVL respectively. Codons were optimized for E. coli and hamster usage. Several overlapping oligonucleotides (69-90 bases in length) extending the full length of the hVL and hVH with two short flanking primers for each chain were used to separately synthesize the two genes by recursive PCR essentially as described in Prodromou et al, (1992) Protein Eng 5(8): 827-9. Resulting D ⁇ A fragments of the correct length were gel purified and then cloned into an E. coli bicistronic expression plasmid (ampicillin resistant).
  • the amino acid sequences of the variable regions of the CDR-grafted antibody (also termed the "template"), termed 8L2-4D5, are also shown in Figures 1 A and IB.
  • the affinity of 8L2-4D5 was determined using BIAcore analysis as described above. 8L2-4D5 bound human NGF with a K D of approximately 38 nM.
  • V71K substitution was introduced into the CDR-grafted heavy chain using recombinant PCR site directed mutagenesis as described in Innis et al, (1995) PCR strategies. San Diego, Academic Press. This substitution replaced the human framework residue with the corresponding mouse framework residue.
  • the resulting antibody was termed 8L2-6D5, and the amino acid sequence of the heavy chain variable region of 8L2-6D5 is shown in Figure 1 A.
  • the affinity of 8L2-6D5 was determined using BIAcore analysis as described above.
  • the Fab fragment of 8L2-6D5 bound human NGF with a Kd of approximately 15 nM. 8L2-6D5 was chosen as template for affinity maturation.
  • CDRs LI, L2, HI and H2 were subjected to humanization and affinity maturation. Amino acid positions in CDRs LI, L2, HI, and H2 were identified that are not essential for the structure of the CDRs based on the Chothia canonical structure (see Al-Lazikani et al (1997) J. Mol. Biol. 273(4):927-48); and subjected to randomization as follows. Two libraries were prepared containing the light chain mutations or heavy chain mutations shown in Table 2, and the grafted (mouse) CDR L3 or CDR H3, respectively, using PCR cassette mutagenesis with degenerate oligonucleotides as described in Kay et al.
  • N35 was changed to N
  • T, S, or Y CDR-H2 M50 was changed to all 20 natural amino acids
  • H32 was changed to H, N, K, D, E, Q, or Y CDR-L2
  • Y50 was changed to Y, D, A, or S 151 was changed to I, T, A, or V F54 was changed to F or L S56 was changed to S and T
  • each library was further paired with the corresponding CDR-grafted light or heavy chain (for example, the H1/H2 library was paired with CDR-grafted light chain), the antibody was expressed, and affinity to human NGF of the individual clones was screened using the BIACORE surface plasmon resonance (SPR) system (BIAcore, Inc. Piscataway, NJ) according to the manufacturer's instructions and as described above, koff, kon and K D were determined.
  • SPR surface plasmon resonance
  • Antibody clones were ranked based on k 0ff rates, since generally most variation in affinity is seen in k off rates, and further because k 0f r rates are independent of antibody concentration.
  • Table 3 LI and L2 amino acid sequences, HI and H2 amino acid sequences, and kinetic data for clones that bound following affinity screening of H1/H2 or L1/L2 library clones.
  • M50 M, I, G, Q, S, L bound.
  • S26 S, and F bound.
  • D28 D, S, A, Y bound.
  • H32 H, N, Q bound.
  • Y50 Y bound. 151: I, T, V, A, bound.
  • F54 F bound S56: S and T bound
  • CDR-L2 Y50Y (no change); 15 IT; F54F (no change); S56S (no change) [0353]
  • H19-L129 a single clone, termed H19-L129, which also included the grafted H3 and L3 CDRs.
  • the sequence of the heavy chain and light chain variable regions of H19-L129 is shown in Figures IA and IB, and Table 4 shows the amino acid sequence of CDRs LI, L2, HI and H2.
  • H19-L129 bound NGF with a KD of approximately 1 nM, as determined using BIAcore analysis as described herein.
  • Table 4 Amino acid sequence of CDRs HI, H2, LI and L2 and kinetic data for combined clone H19-L129.
  • H3 and L3 CDRs was carried out in two steps.
  • library scanning mutagenesis each amino acid residue in H3 and L3 was individually prescreened in order to identify amino acid positions at which a mutation resulted in increased binding affinity to human NGF.
  • small library randomization analysis also termed "small library randomization analysis”
  • a subset of amino acid positions in H3 and L3 were selected for preparation of the affinity maturation library, and the affinity maturation library was screened for affinity to human NGF using BIAcore analysis as described herein. It is appreciated that these techniques can be generally applied.
  • Each amino acid position in the H3 and L3 CDRs was individually prescreened for substitutions which resulted in increased binding affinity to human NGF.
  • the frequency of amino acid substitutions at any given position that resulted in improved binding, the same binding, worse binding or no binding provided information relating to relating to positions in the CDRs that can be changed to many different amino acid (including all 20 amino acids), and positions in the CDRs which cannot be changed or which can only be changed to a few amino acids.
  • Amino acid substitutions resulting in increased binding affinity were also identified. Based on the results of this screening, a subset of amino acid positions in CDRs H3 and L3 were selected for preparation of an affinity maturation library.
  • H3 CDRs was randomized to all 20 amino acids, one at a time, resulting in several (5 libraries for the light chain and 13 libraries for the heavy chain) small libraries, each with a complexity of 20 amino acid possibilities at each amino acid position. In all cases, the native (i.e., unchanged) amino acid was represented in the library. Libraries were prepared by PCR cassette mutagenesis with degenerate oligonucleotides as described in Kay et al. (1996), Phage display of Peptides and Proteins: a laboratory manual, San Diego, Academic Press, using the doping codon NNK to randomize one amino acid position to include 20 possible amino acids.
  • each member of a library contained a CDR3 (either H3 or L3) with one amino acid substitution, and 5 grafted CDRs.
  • 20-80 clones from each small library were screened using BIAcore analysis as described herein.
  • This library combined mutations in H3 and L3 at the same time in CDR- grafted clone 8L2-6D5, and separately in the background of H19-L129, and had a diversity of 80 different clones.
  • Table 7 shows the amino acid residues selected for substitution and the amino acids that were substituted at each position.
  • YlOl was changed to Y and , C. (Note that C was included because use of codon TRS in one degenerated oligonucleotide also generated codon C).
  • G103 was changed to A ,P, S CDR-L3:
  • K92 was changed to all twenty amino acids. A, R, K, and H bound.
  • E3 (interchangeably termed "3E") and 3C, were selected for further characterization.
  • E3 comprised the following CDR substitutions: CDR-H3: YlOl W, G103A; and CDR-L3: S91E, K92H, which were combined into a single clone which also included the following LI, L2, HI and H2 mutations:
  • CDR-L2 15 IT.
  • the sequence of the heavy chain and light chain variable regions of E3 is shown in Figures IA and IB.
  • 3C comprised the following CDR substitutions: CDR-L3: S91E; K92R; CDRH3: Y101W; G103A, which were combined into a single clone which also included the LI, L2, HI and H2 mutations described for clone 3E.
  • 3E and 3C sequences were cloned into mammalian expression vectors for production of Fab and full antibody, and expressed in HEK293 cells and purified using Ni- NTA or protein A chromatography. Pure protein was accurately quantified by amino acid analysis.
  • mouse antibody 911 bound NGF with a KD of 3.7 nM, k off of 8.4x10 "5 s and ko chorus of2.2xl0 4 Ms "1 .
  • Antibody E3 (interchangeably termed 3E) was selected for further analysis based on the high binding affinity.
  • 3E Antibody E3
  • 2.5 nM of human NGF was premixed and incubated for one hour with 0 to 50 nM of antibody E3 (Fab). After the incubation, samples were injected at 10 ul/minute on a BIAcore CM5 chip containing 260 RU of p75 (channel 2) and 600 RU of trkA (channel 3), and percent binding was determined. The results of this analysis are shown in Figure 11.
  • Example 2 evaluation ofNGF-blocking ability of anti-NGF antibodies using mouse El 3.5 trigeminal neuron survival assay
  • the ability of Fab E3 or full antibody E3 to block NGF activity was evaluated by measurement of the capacity of the antibody to inhibit NGF-dependent survival of mouse E13.5 trigeminal neurons in vitro.
  • the trigeminal ganglion is comprised of cutaneous sensory neurons that innervate the facial region.
  • the survival of mouse El 3.5 trigeminal neurons is a sensitive assay to evaluate the NGF-blocking activity of anti-NGF antagonist antibodies because NGF is required to support survival of these neurons. For example, at saturating concentrations of NGF, the survival is close to 100% by 48 hours in culture. By contrast, less than 5% of the neurons survive by 48 hours in absence of NGF.
  • the survival assay was conducted as follows: time-mated pregnant Swiss
  • Webster female mice were euthanised by CO2 inhalation.
  • the uterine horns were removed and the embryos at embryonic stage El 3.5 were extracted and decapitated.
  • the trigeminal ganglia were dissected using electrolytically sharpened tungsten needles.
  • the ganglia were then trypsinized, mechanically dissociated and plated at a density of 200-300 cells per well in defined, serum-free medium in 96-well plates coated with poly-L-ornithine and laminin.
  • anti-NGF Fabs or antibodies were assessed by adding to the trigeminal neurons varying doses of anti-NGF antibodies Mab 911 (Fab), 8L2-6D5; H19-L129; E3 and 3C; and human or rat NGF at the following concentrations: 0.4 ng/ml ( ⁇ 15 pM; this concentration represented a saturating concentration of NGF for survival) and 0.04 ng/ml ( ⁇ 1.5 pM; this concentration is around the IC50).
  • the cells were subjected to an automated immunocytochemistry protocol performed on a Biomek FX liquid handling workstation (Beckman Coulter) as follows: fixation using 4% formaldehyde, 5% sucrose, and PBS; permeabilization using 0.3% Triton X-100 in PBS); blocking of unspecific binding sites using 5% normal goat serum, 0.11% BS A in PBS; and sequential incubation with a primary and secondary antibodies to detect neurons.
  • the primary antibody was rabbit polyclonal antibody against the protein gene product 89.5 (PGP9.5, Chemicon), an established neuronal phenotypic marker.
  • the secondary antibody was Alexa Fluor 488 goat anti-rabbit (Molecular Probes), together with the nuclear dye Hoechst 33342 (Molecular Probes) to label the nuclei of all the cells present in the culture.
  • Image acquisition and image analysis were performed on a Disco very-I/Genll Imager (Universal Imaging Corporation). Images were automatically acquired at two wavelengths for Alexa Fluor 488 and Hoechst 33342, with the nuclear staining being used as reference point for the image-based auto-focus system of the Imager, since nuclear staining is present in all of the wells. Appropriate objectives and number of sites imaged per well were selected to cover the entire surface of each well.
  • Figure 5 is a graph comparing the NGF blocking effect of various Fabs in the presence of either 0.04 ng/ml of human NGF (approximately 1.5 pM; shown in the lower panel) or 0.4 ng/ml human NGF (approximately 15 pM; shown in the upper panel). 1.5 pM of NGF was around the EC50 of NGF promoting survival, while 15 pM represented a saturating concentration of NGF. Survival of E13.5 mouse trigeminal neurons in various concentrations of Fab E3; murine 911 Fab; and Fab H19-L129 and Fab 8L2-6D5 was assessed as described above.
  • the IC50 (in pM) was calculated for each Fab at each NGF concentration, and is shown in Table 9.
  • Fab E3 strongly blocked human NGF-dependent trigeminal neuron survival, with an IC50 of approximately 21 pM in the presence of 15 pM human NGF, and an IC50 of approximately 1.2 pM in the presence of 1.5 pM human NGF.
  • Fabs 3C and H19-L129 also strongly blocked human NGF-dependent trigeminal neuron survival.
  • Figure 6 is a graph comparing the NGF blocking effect of various Fabs in the presence of either 0.04 ng/ml of rat NGF (approximately 1.5 pM; shown in the lower panel) or 0.4 ng/ml rat NGF (approximately 15 pM; shown in the upper panel). 1.5 pM of NGF was around the EC50, while 15 pM represented a saturating concentration of NGF. Survival of El 3.5 mouse trigeminal neurons in various concentrations of Fab E3; murine Fab 911; and Fab H19-L129 and 8L2-6D5 was assessed as described above. The EC50 (in pM) was calculated for each Fab at each NGF concentration, and is shown in Table 9.
  • Fab E3 strongly blocked human NGF-dependent trigeminal neuron survival, with an IC50 of approximately 31.6 pM in the presence of 15 pM rat NGF, and an IC50 of approximately 1.3 pM in the presence of 1.5 pM rat NGF.
  • Fabs 3C and H19-L129 also strongly blocked rat NGF-dependent trigeminal neuron survival.
  • Example 3 Evaluation of the specificity of anti-NGF antibody E3 using mouse trigeminal and nodose neuron survival assays
  • the ability of antibody E3 to specifically block NGF activity was evaluated by measurement of the capacity of the antibody to inhibit survival of mouse El 7/ 18 trigeminal neurons in vitro in the presence of saturating concentrations of NGF, the NGF- related neurotrophin NT3, or the NGF-unrelated neurotrophic factor, macrophage stimulating protein (MSP).
  • MSP macrophage stimulating protein
  • the survival of mouse El 7/18 trigeminal neurons is a sensitive assay to evaluate the NGF-blocking activity of anti-NGF antagonist antibodies because NGF is required to support survival of these neurons at higher concentrations than the level ' of NGF required to support survival of El 3.5 TG neurons).
  • Webster female mice were euthanised by CO2 inhalation.
  • the uterine horns were removed and the embryos (at embryonic day 17 or 18) were extracted and decapitated.
  • the trigeminal and nodose ganglia were dissected and cleaned.
  • the ganglia were then trypsinised, mechanically dissociated and plated at a density of 100-300 cells per well in defined, serum-free medium in 4-well plates (Greiner) coated with poly-L-ornithine and laminin.
  • El 7/18 trigeminal neurons were grown either without added neurotrophic factors (negative control) or in the presence of saturating concentrations of human NGF (400pM and 15 ⁇ M) (positive control); NT3 (400 pM); or MSP (600pM).
  • Duplicate cultures were set up that included varying concentrations of E3 and 911 Fabs and full antibodies. Concentration of Fab and full antibodies was indicated per binding site (e.g., a full antibody contains two binding sites, while a Fab contains one binding site).
  • El 7 nodose neurons were grown either in the absence of added neurotrophic factors (negative control), or with saturating concentrations of BDNF (400pM) (positive control) or NT4/5 (400pM) or NGF unrelated growth factor ILF (interleukin inhibitory factor). High concentrations of neurotrophins were used, as the goal of this experiment was to test specificity of the antibodies. Duplicate cultures were set up that included varying again with and without the addition of antibodies E3 and 911. After 48 hours in culture the total number of neurons surviving in each well under each condition was ascertained by manual counting using a phase-contrast microscope.
  • Figures 14-15 are graphs showing that anti- NGF antagonist antibody E3 or Fab E3 did not inhibit the survival promoted by NT3, and MSP, even at antibody concentrations as high as 200 nM. By contrast, 20 nM of antibody E3 or Fab 3E and Fab 911 totally blocked NGF-elicited survival. Mouse anti-NGF antagonist antibody 911 was also tested, and similar results were observed.
  • Figure 14 is a graph showing comparison of the effect of various concentrations (20 nM, 2 nM, or 0.2 nM) of E3 Fab (termed “3E” in the figure) and mousp antibody 911 Fab on survival of El 8 trigeminal neurons in the presence of no added neurotrophin (termed “control”), 400 pM NGF (termed “NGF-400pM), 10 nM NT3 (termed “NT3-10nM) or 600 pM MSP (termed "MSP-600 pM).
  • Figure 15 is a graph depicting comparison of the effect of various concentrations (200 nM and 80 nM) of E3 Fab and full antibody and mouse antibody 911 full antibody and Fab of survival of El 7 trigeminal neurons in the presence of no added neurotrophins (termed “no factor”), 400 pMNGF (termed “NGF-400pM), 10 nM NT3 (termed “NT3-10nM) or 600 pM MSP (termed "MSP-600 pM).
  • Figure 16-17 are graphs showing that anti-NGF antagonist antibody E3 or
  • Figure 16 is a graph showing comparison of the effect of various concentrations (200 nM or 80 nM) of full antibody E3 (termed “3E in the figure"), Fab E3, full antibody 911, or Fab 911 on the survival of El 7 nodose neurons in the presence of no added neurotrophins (termed "no factors"), 400 pM BDNF (termed “BDNF-400pM), 400 pM NT4/5 (termed “NT4/5-400pM), or 2.5 nM LIF (termed "LIP-2.5 nM).
  • Figure 17 is a graph showing comparison of the effect of various concentrations (200 nM, 20 nM, 2nM) of Fab E3 (termed “3E in the figure"), or Fab 911 on the survival of El 7 nodose neurons in the presence of no added neurotrophins (termed “control”), 400 pM BDNF (termed “BDNF-400pM), 400 pM NT4/5 (termed “NT4/5-400pM), or 2.5 nM LIF (termed "LIP-2.5 nM).
  • Example 5 Preparation of mammalian expression vectors and expression of antibody E3 in mammalian cells
  • Vector Db.911.3E is an expression vector comprising the heavy chain variable region of the E3 antibody and the human IgG2a constant region, and is suitable for transient or stable expression of the heavy chain.
  • Db.911.3E consists of nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 1-612); a synthetic intron (nucleotides 619-1507); the DHFR coding region (nucleotides 707-1267); human growth hormone signal peptide (nucleotides 1525-
  • Vector Eb.911.3E is an expression vector comprising the light chain variable region of the E3 antibody and the human kappa chain constant region, and is suitable for transient expression of the light chain.
  • Eb.911.3E consists of nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); human growth hormone signal peptide (nucleotides 1173-1150); antibody E3 light chain variable region (nucleotides 1251-1571); human kappa chain constant region (nucleotides 1572-1892); SV40 late polyadenylation signal (nucleotides 1910-2153); SV40 enhancer region (nucleotides 2154-2399); phage fl region (nucleotides 2487-2942) and beta lactamase (AmpR) coding region (nucleotides 3379
  • Vector Eb.pur.911.3E is an expression -vector comprising the light chain variable region of the E3 antibody and the human kappa constant region, and is suitable for stable expression of the light chain.
  • Eb.pur.911.3E consists of nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 1-612); human EF-1 intron (nucleotides 619-1758); pac gene (puromycinR) coding region (nucleotides 739-1235); human hsp70 5'UTR region (nucleotides 1771- 1973); human growth hormone signal peptide (nucleotides 1985-2062); antibody E3 light chain variable region (nucleotides 2063-2383); human kappa chain constant region (nucleotides 2384-2704); SV40 late polyadenylation signal (nucleotides 2722-2965); SV40 enhancer region (nucleotides 2966-3211); phage fl region (nucleotides 3299-3654) and beta lactamase (AmpR) coding region (nucleotides 41
  • Eb.pur.911.E3 was deposited at the ATCC on January 8, 2003, and was assigned ATCC Accession No. PTA- 4894.
  • Transient cell expression was perfomed as follows: CHO and HEK293T cells in 150 mm dishes were transiently co-transfected with 25 ug of each plasmid (i.e., one plasmid containing the heavy chain and one plasmid containing the light chain). DNA was mixed with 100 ul lipofectamine 2000 (Invifrogen) according to the manufacturer's instructions. The DNA-lipid complexes were allowed to contact the cells in DMEM/F12 medium without serum or antibiotics for 5 hours.
  • Anti-NGF antibody E3 is effective in treating post-surgical pain [0391]
  • Antibody E3 comprised the human heavy chain IgG2a constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype IgG2a sequence; see Eur. J. Immunol. (1999) 29:2613-2624); the human light chain kappa constant region; and the heavy and light chain variable regions shown in Tables IA and IB.
  • Bleeding was controlled throughout surgery by pressure applied through a gauze pad.
  • the wound was closed with two mattress sutures (5-0 ethilon black monofilament). These sutures were knotted 5-6 times, with the first knot loosely tied.
  • the wound site was swabbed with bacitracin solution. Animals were allowed to recover and rest in clean cages for two hours or more before behavioral testing began.
  • Weight bearing was a good correlate of how willing the animal was to use the limb, and therefore was an effective measure of pain relief.
  • the E3 antibody was injected intra peritoneal (i.p.) at various concentrations of the antibody (0.004, 0.01, 0.02, 0.1, 0.6, and 1 mg per kilogram of animal weight) at 15 hours pre-incision.
  • the negative control group received no antibody but was injected i.p. with a saline solution.
  • Fentanyl at 0.01 mg/kg was injected i.p. as a positive control 30 minutes before testing at 24 hours post-surgery.
  • Example 7 Assessment of analgesic effects of anti-NGF antagonist antibody 911 in a rat model of rheumatoid arthritis
  • the nociceptive intensity was evaluated by gentle flexion of the hindpaw 24 hours later, and the intensity of the level of vocalization was recorded for each animal.
  • group 4 indomethacin
  • D18 the nociceptive intensity was evaluated by gentle flexion of the hindpaw one hour after oral administration of indomethacin (10 mg/kg).
  • the intensity of the level of vocalization was also recorded for each animal.
  • the test substances were administered in a blind and random manner by intravenous route under a volume of 5 ml/kg, whereas indomethacin was administered by oral route under a volume of 10 ml/kg.
  • 911 (10 mg/kg) was intravenously administered at D14 or D17 and pain measurement was performed at D18.
  • Indomethacin (10 mg/kg) was orally given at D18 and pain measurement was performed one hour after dosing.
  • anti-NGF antibody 911 significantly reduced pain in a rat model of rheumatoid arthritis 24 hours or 4 days after a single administration of the antibody.
  • Example 8 Pharmacological effects of anti-NGF antagonist antibody E3 and 911 in a rat model of rheumatoid arthritis
  • NGF antagonist antibody E3 and 911 were investigated in a model of complete Freund's adjuvant (CFA)-induced chronic arthritis in rats in comparison with indomethacin used as an internal positive control substance.
  • Analgesic effects of E3 and 911 were evaluated by the measurement of nociceptive response.
  • -Anti-infl-immatory effects were evaluated by paw volume, arthritis index (inflammation score), body and hindpaws weight.
  • Paw cytokine levels IL-6, IL-l ⁇ , TNF- ⁇ and TGF- ⁇ l
  • circulating TGF- ⁇ l in serum E3 and 911 plasma concentrations
  • biological parameters and X-ray radiographies were performed at the end of experiment.
  • Group 4 arthritic rats / E3, 1 mg/kg , i.v.
  • E3 and 911 were extemporaneously prepared in saline from the stock solution to the desired concentration.
  • osmolarity (mosm/L) for saline E3, and 911 were 278, 269, and 308 respectively; pH for saline, E3, and 911 were 5.93, 6.76, 6.71 respectively.
  • osmolarity (mosm/L) for saline E3, and 911 were 280, 270, and 309 respectively; pH for saline, E3, and 911 were 5.86, 6.72, and 6.59 respectively.
  • E3 or 911 or saline were administered by i.v. bolus injection on Day 14 and
  • a group of 10 rats did not receive any intradermal injection (non arthritic rats).
  • the arthritic rats were included in the study using the following criteria: all included rats displayed an increase of mean paw volume (mean of the left and right paw volume) of at least 0.30 ml compared to the mean paw volume (mean of the left and right paw volume) in the non arthritic group (paw volume measurement as described below); all included rats displayed a vocalization upon gentle flexion (nociceptive response measurement as described below); and all included rats displayed a score of arthritis index of 2-3 on each hindpaw (arthritis index measurement as described below) (the animals with a score of 0, 1 or 4 were discarded). 3. Body weight
  • the right and left hindpaw volume of each rat was measured using a plethysmometer. The measurements were performed at the following times (after induction of arthritis): Day 14 (before i.v. bolus or p.o. administration); and Day 24 (5 days after the last i.v. bolus injection or 24 h after the last p.o. administration). All measurements were performed between 9:00 and 12:00 am. All the data were collected and stored by the WinDas software.
  • the nociceptive response was evaluated by gentle flexion of the right and left hindpaw repeatedly 2 times at intervals of 4 to 5 sec with a finger of the operator (arthritic rats). The intensity of the level of vocalization was recorded for each animal for each hindpaw (2 times: on right hindpaw: si and s3; 2 times: on left hindpaw: s2 and s4). This evaluation was performed at the following times (after induction of arthritis): Day 14 (before i.v. bolus or p.o. administration); Day 18 (before the second i.v. bolus injection or 1 hr after p.o. administration); and Day 24 (5 days after the last i.v. bolus injection or 24 h after the last p.o. administration). All measurements were performed between 9:00 and 12:00 am except at D 14 (7:30 - 9:00 am) and D 24 (7:30 - 9: 00 am).
  • Measurement ofE3 or 911 concentration (groups 2, 4 and 5): A part of blood sample was collected in tubes containing Li-Heparin (maintained on ice) and centrifuged at 2500-3000 g for 10 min. Plasma samples (at least 100 ⁇ L) were obtained, frozen in liquid nitrogen, stored at -80°C. One sample was slightly hemolyzed (vehicle- treated arthritic rat # 36).
  • TGF- ⁇ l levels were measured using human TGF- ⁇ l ELISA kit (ref. DB100, Batch 212258 and 213610, R&D Systems - France). [0416] Blood collection for hematological parameters (groups 1-2-3-4-5: 50 vials):
  • a part of blood sample was collected in tubes containing K3 - EDTA (at least 100 ⁇ L). The determination of parameters were performed on the day of the collection and the samples were not stored. The hematological parameters including red blood cells, white blood cells, platelets, hemoglobin, hematocrit were measured with a hematology cell counter (D 24). Some hematological parameters were not measured due to the clotted samples (vehicle- treated non arthritic rat: # 10; E3-treated arthritic rats: # 59, # 67; 911-treated arthritic rats: # 16).
  • Cytokine levels measurement Cytokine levels of TNF- ⁇ (Rat TNF- ⁇ ELISA kit, ref. RTA00, Batch 213718, R&D Systems, France) , IL-l ⁇ Rat IL-l ⁇ ELISA kit, ref. RLBOO, Batch 212435, R&D Systems, France), IL-6 Rat IL-6 ELISA kit, ref. R6000, Batch 211773, 214008 and 214362, R&D Systems, France), and TGF- ⁇ lHu an TGF- ⁇ l ELISA kit, ref. DB100, Batch 212258 and 213610, R&D Systems, France) were determined in duplicate, according to the manufacturer's procedure. Aliquots of hind paw homogenates were stored at -80°C.
  • X-ray radiographys hindpaws
  • X-ray analysis was focused on articular erosions, articular space, periosteum abnormalities on both hindpaws. All the radiographies were analyzed by looking at seven different items: the soft tissue damage, deformity, demineralization, joint space, erosions, osteogenesis and periostal reaction.
  • the first six items were analyzed independently by looking at the worse hind foot.
  • the periostal reaction was analyzed by looking at the tail. For each item, the score goes from 0 (normal) to 4 (maximal damage). Therefore the total score goes from 0 to 28.
  • the radiographic interpretation was done by the same reader without knowing anything about the animals (treated or not treated).
  • Hindpaws (left and right) weight was expressed in grams. Cytokine levels (IL-6, IL-l ⁇ , TNF- ⁇ and TGF- ⁇ l) of each hind paw was expressed in pg/ml. Circulating levels of TGF- ⁇ l was expressed in pg/ml. Radiological index for each parameter (demineralization, erosions, periostal reaction, soft tissue damage, space joint, osteogenesis deformity) and total radiological index (total score) were calculated from the sum of the scores obtained for each parameter.
  • E3 (1 mg/kg i.v. at D 14 and D 19) strongly and significantly decreased the nociceptive response by about -4167 mV (% inhibition : 79 %) and -5905 mV (% inliibition: 100 %) at D 18 and D 24, respectively compared to the vehicle-treated arthritic group (D 18: 1112 ⁇ 401 vs 5279 ⁇ 326 mV; D 24: 0 ⁇ 0 vs 5905 ⁇ 345 mV). 911 (10 mg/kg i.v.
  • E3 treatment (1 mg/kg i.v. at D 14 and D 19)
  • the body weight moderately and significantly increased from D 17 to D 24 with a maximum of about 46 g at D 24 compared to the vehicle-treated arthritic group (264 ⁇ 5 g 5218 ⁇ 3 g).
  • 911 treatment (10 mg/kg i.v. at D 14 and D 19) the body weight moderately and significantly increased from D 18 to D 24 with a maximum of about 47g at D 24 compared to the vehicle-treated arthritic (265 ⁇ 7 vs 218 ⁇ 3 g).
  • Paw volume [0425] On D 14, a randomization was performed in order to obtain homogenous groups in terms of paw volume. As shown in Table 13, on D 14, the hindpaw volume (mean of the right and left paw volume) was significantly greater in arthritic group than that in non arthritic group (2.10 ⁇ 0.05 vs 1.44 ⁇ 0.02 mL (Student t test P ⁇ 0.05)). Indomethacin (3 mg/kg/day p.o. for 10 days) significantly decreased the paw volume by about -0.75 mL (D 24) compared to the vehicle-treated arthritic group (1.59 ⁇ 0.03 mL vs 2.34 ⁇ 0.08 mL). E3 (1 mg/kg i.v.
  • cytokines levels of arthritic group were similar in left and right paw: 259.7 ⁇ 38.5 vs 219.2 ⁇ 32.4, 4802.8 ⁇ 365.5 vs 4007.1 ⁇ 380.4, 17.8 ⁇ 1.6 TO 18.6 ⁇ 1.9 and 9735.0 ⁇ 1219.8 vs 9161.4 ⁇ 846.1 pg/ml for IL-6, IL-l ⁇ , TNF- ⁇ and TGF- ⁇ l respectively. Indomethacin slightly, but significantly, decreased the TGF- ⁇ l level in right paw after 3 mg/kg/day p.o.
  • n 10 animals per group except for Non-arthritic/vehicle (Right paw), Arthritic/vehicle
  • Hematological parameters As shown in Table 17, the hematological parameters such as white blood cells and platelets were greater in vehicle-treated arthritic rats in comparison to vehicle- treated non arthritic rats (Student t test P ⁇ 0.05), whereas the red blood cells, hemoglobin and hematocrit (Student t test P>0.05) were unchanged. Indomethacin did not affect the blood parameters after 3 mg/kg/day p.o. (for 10 days) compared to the vehicle-treated arthritic group. E3 (1 mg/kg i.v. on D 14 and D 19) did not affect the blood parameters compared to the vehicle-treated arthritic group. 911 (10 mg/kg i.v. on D 14 and D 19) did not affect the blood parameters compared to the vehicle-freated arthritic group.
  • Hindpaw weight As shown in Table 18, the left and right hindpaw weight was greater in vehicle-treated arthritic rats than in vehicle-treated non arthritic rats (3.43 ⁇ 0.11 vs 1.98 ⁇ 0.01 and 3.32 ⁇ 0.12 TO 1.99 ⁇ 0.02 g, respectively) (Student t test or Mann-Withney P ⁇ 0.05). Indomethacin significantly decreased the hindpaws weight after 3 mg/kg/day p.o. (for 10 days) compared to the vehicle-freated arthritic group (left hindpaw: 2.23 ⁇ 0.04 vs 3 A3 ⁇ 0.11 g; right hindpaw: 2.20 ⁇ 0.05 vs 3.32 ⁇ 0.12 g).
  • E3 (1 mg/kg i.v. on D 14 and D 19) only significantly increased the left hindpaw weight compared to the vehicle-treated arthritic group (left hindpaw: 3.86 ⁇ 0.14 TO 3.43 ⁇ 0.11 g; right hindpaw: 3.72 ⁇ 0.13 TO 3.32 ⁇ 0.12 g).
  • 911 (10 mg/kg i.v. on D 14 and D 19) only significantly increased the right hindpaw weight compared to the vehicle-treated arthritic group (left hindpaw: 3.73 ⁇ 0.12 TO 3.43 ⁇ 0.11 g; right hindpaw: 3.83 ⁇ 0.15 r ⁇ 3.32 ⁇ 0.12 g).
  • Table 18 Effects of E3 and 911 after i.v. injection (2 days on D 14 and D 19) on hindpaws weight in rheumatoid arthritis in rats (Measurement at D 24)
  • X-ray analysis As shown in Table 19, a total score of 0.0 ⁇ 0.0 was observed in the vehicle- treated non arthritic rats.
  • the vehicle-treated arthritic rats have a total score of 15.1 ⁇ 1.3 with high scores for demineralization (2.4 ⁇ 0.3), erosions (2.7 ⁇ 0.3), soft tissue damage (3.1 ⁇ 0.2) and space joint (3.3 -fc 0.2), a moderate score for periostal reaction (1.0 ⁇ 0.3), osteogenesis (0.8 ⁇ 0.2) and deformity (1.8 ⁇ 0.2).
  • Indomethacin (3 mg/kg/day p.o.
  • Example 9 Effects of different doses of anti-NGF antibody E3 in a rat model of rheumatoid arthritis
  • E3 The ability of E3 to produce reduction in pain in arthritic rats was further investigated by examining the dose response relationship between E3 administration and pain reduction. Rats were treated with adjuvant to induce arthritis as described above. Ten rats not injected with adjuvant were used as non-arthritic controls. Fourteen days after adjuvant injection, animals were qualified into the study based on the criteria stated above, randomized into eight groups often rats and tested for the intensity of their vocalization response. They were then dosed on day 14 with saline, or 0.003 mg/kg, 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg or 5 mg/kg of E3 antibody as described above.
  • mice were tested for their vocalization response on days 16, 18, 20, and 24. Animals were redosed with saline or the same dose of E3 on day 18 after the vocalization testing. Animals were also weighed each day, starting at day 14. Thus, animals were dosed twice with a given dose of antibody or saline on days 14 and 18, and assessed for pain five times, on days 14, 16, 18, 20 and 24. Data are shown in Tables 20-22 and in Figures 20-22.
  • Vocalization intensity values are expressed in mV as mean ⁇ s.e.m.
  • Non-Arthritic vehicle 0.003 mg/kg 0.01 mg/kg 0.03 mg/kg
  • Non-Arthritic vehicle 0.003 mg/kg 0.01 mg/kg 0.03 mg/kg
  • rats treated with E3 showed less apparent weight loss than saline treated arthritic rats (Table 22 and Figure 22).
  • rats treated with high doses of antibody E3 were recovering the earlier weight loss, and actually gaining weight faster than their non-arthritic cohorts (Table 21 and Figure 21).
  • Vector Eb.911.3E is a polynucleotide encoding the E3 light chain variable region; vector Eb.pur.911.3E is a polynucleotide encoding E3 light chain variable region, and vector Db.911.3E is a polynucleotide encoding the E3 heavy chain variable region.
  • Heavy chain variable region (Kabat CDRs are underlined; Chothia CDRs are BOLD AND ITALICIZED)
  • E3 heavy chain extended CDRs CDRHl GFSLIGYDLN (SEQ ID NO:3)
  • CDRL1 RASQSISNNLN (SEQ ID NO:6)
  • CDRL2 YTSRFHS (SEQ ID NO:7)
  • CDRL3 QQEHTLPYT (SEQ ID NO:8)
  • CDRH2 MIWGDGTTDYNSALKS (SEQ ID NO: 10)
  • CDRL1 RASQDISNHLN (SEQ ID NO: 12)
  • CDRL2 YISRFHS (SEQ ID NO: 13)
  • CDRL3 QQSKTLPYT (SEQ ID NO: 14)

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AU2003299898A AU2003299898B2 (en) 2002-12-24 2003-12-24 Anti-NGF antibodies and methods using same
BRPI0317738A BRPI0317738B8 (pt) 2002-12-24 2003-12-24 anticorpos anti-ngf ou fragmentos dos mesmos, seu método de fabricação, uso, polinucleotídeo, vetor e micro-organismo transgênico, bem como composição farmacêutica e kit compreendendo os referidos anticorpos ou fragmentos
EP10176615.2A EP2263692B1 (en) 2002-12-24 2003-12-24 Anti-NGF antibodies and methods using same
KR1020127025397A KR101410692B1 (ko) 2002-12-24 2003-12-24 항-ｎｇｆ 항체 및 그것을 이용하는 방법
IL169221A IL169221A (en) 2002-12-24 2005-06-16 Antibodies to NGF and methods of using them
NO20053583A NO336655B1 (no) 2002-12-24 2005-07-22 Anti-nervevekstfaktor (NGF)-antistoff, eller fragment derav, farmasøytisk preparat og sett derav, fremgangsmåter for deres fremstilling, isolert polynukleotid, vektor og isolert vertscelle og anvendelse derav for behandling av smerte, spesielt smerte forbundet med osteoartritt.
HK05110620.2A HK1076251A1 (en) 2002-12-24 2005-11-23 Anti-ngf antibodies and methods using same
HK08101386.2A HK1110513A1 (en) 2002-12-24 2008-02-05 Anti-ngf antibodies and methods using same
AU2011201157A AU2011201157C1 (en) 2002-12-24 2011-03-16 Anti-NGF antibodies and methods using same
IL218087A IL218087B (en) 2002-12-24 2012-02-13 Anti-ngf antibodies and methods of using them
NO20150469A NO339595B1 (no) 2002-12-24 2015-04-20 Anti-nervevekstfaktor (NGF) antagonist antistoff eller antigenbindende fragment derav for anvendelse ved behandling av osteoartritt-smerte i et individ samt for anvendelse ved fremstilling av et medikament for behandling av osteoartritt-smerte i et individ.
IL263573A IL263573A (en) 2002-12-24 2018-12-09 Anti-ngf antibodies and methods of using them

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