WO2016008851A1 - Anticorps anti-il-1b - Google Patents

Anticorps anti-il-1b Download PDF

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Publication number
WO2016008851A1
WO2016008851A1 PCT/EP2015/065979 EP2015065979W WO2016008851A1 WO 2016008851 A1 WO2016008851 A1 WO 2016008851A1 EP 2015065979 W EP2015065979 W EP 2015065979W WO 2016008851 A1 WO2016008851 A1 WO 2016008851A1
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seq
amino acid
acid sequence
antibody
chain variable
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PCT/EP2015/065979
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English (en)
Inventor
Klaus Fuchs
Peter Fischer
John Edward Park
Daniel Peter
Daniel SEELIGER
Bernd Weigle
David Michael Wyatt
Keith A. Canada
Pankaj Gupta
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Boehringer Ingelheim International Gmbh
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Publication of WO2016008851A1 publication Critical patent/WO2016008851A1/fr

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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/245—IL-1
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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

  • This invention generally relates to anti-IL-1 B antibodies for diagnostic and therapeutic use. More specifically, optimized anti-IL-1 B antibodies and methods of use for the treatment of various diseases or disorders are disclosed. Pharmaceutical compositions and kits comprising such compounds are also disclosed. Background of the Invention
  • IL-1 B lnterleukin-1 beta
  • catabolin a cytokine protein.
  • IL-1 B is a member of the interleukin 1 family of cytokines and is also described as interleukin 1 , beta; IL-1 b; IL-1 ⁇ ; IL1 -BETA; IL1 F2. This cytokine is produced by activated
  • IL-1 B precursor comprises 269 amino acids (NCBI Reference Sequence: NP_000567.1 ).
  • IL-1 a and IL-1 B are the most studied members of the interleukin 1 family and bind to the IL-1 receptor (IL-1 R1 ) and activate signaling. They have a natural antagonist IL- 1 Ra (IL-1 receptor antagonist) that regulates IL-1 a and IL-1 B proinflammatory activity by competing with them for binding sites of the receptor.
  • IL-1 B binds to IL1 R1
  • IL-1 B is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell activation and proliferation, differentiation, and apoptosis. IL-1 B has also been associated with respiratory diseases. For example, human bronchial epithelial cells from patients with COPD respond with a greater increase of IL-1 B secretion than those of non-smokers if exposed to cigarette smoke ex vivo (Rusznak et al. (2000) Am. J Respir. Cell Mol. Biol. 23, 530-536). The relative level of IL-1 B versus endogenous antagonists (IL1 Ra, IL1 sRII) was also shown to be increased in plasma of COPD patients (Sapey et al. (2009) J Clin.
  • IL-1 B levels are elevated in induced sputum in a COPD sub-population (Pauwels et al. (201 1 ) Eur. Respir. J. 38, 1019-1028).
  • IL-1 B levels have been found elevated at time of exacerbations in COPD patients (Gessner et al. (2005) Respir Med. 99, 1229-1240, Kythreotis et al. (2009 BMC. Pulm. Med. 9, 1 1 ).
  • IL-1 B is involved in respiratory diseases and the modulation of IL-1 B could provide promising therapies. There is therefore a need for antagonist molecules against IL-1 B with beneficial pharmacological properties, which can be used as therapeutic agents to treat diseases, in particular respiratory diseases in humans.
  • one aim of the present invention is to provide anti-IL-1 B antagonist molecules, in particular anti-IL-1 B antagonist molecules, which have high binding affinity to IL-1 B.
  • a further aim of the present invention is to provide anti-IL-1 B
  • a further aim of the present invention is to provide anti-IL-1 B antagonist molecules, which have high specificity for IL-1 B.
  • a further aim of the present invention is to provide anti-IL-1 B antagonists, which have potent cellular activity.
  • a further aim of the present invention is to provide anti-IL-1 B antagonists, which have favorable biophysical and/or pharmacological properties.
  • a further aim of the present invention is to provide anti-IL-1 B antagonists, which can be produced well, for example in mammalian cells.
  • a further aim of the present invention is to provide anti-IL-1 B antagonists, which can be produced in a form suitable for the modulation of IL-1 B activity in humans.
  • a further aim of the present invention is to provide anti-IL-1 B antagonists, which can be tested clinically in an efficient manner.
  • aims of the present invention include combinations of any of the aims set forth above.
  • an antibody of the present invention addresses the above needs and provides antibodies that bind to the IL-1 B protein.
  • an antibody of the present invention binds to human IL-1 B with high affinity.
  • an antibody of the present invention is capable of potently inhibiting the activity of human IL-1 B and cynomolgus IL-1 B.
  • an antibody of the present invention is capable of inhibiting the biological activity of human IL-1 B over a wide range of concentrations of IL-1 B, for example including high concentrations of IL-1 B, as for example observed in certain COPD patients.
  • an antibody of the present invention may allow for a more effective treatment of patients with elevated titers of IL-1 B with an antibody of the present invention or for increased flexibility in the dosage of an antibody of the present invention to a patient.
  • an antibody of the present invention is capable of potently inhibiting the activity of human IL-1 B and cynomolgus IL-1 B and is capable of inhibiting the biological activity of human IL-1 B over a wide range of concentrations of IL-1 B.
  • an antibody of the present invention binds at high affinity to human IL-1 B, to cynomolgus IL-1 B and to mouse IL-1 B.
  • an antibody of the present invention can inhibit the activity of human IL-1 B, of cynomolgus IL-1 B and of mouse IL-1 B.
  • the present invention provides anti-IL-1 B antibodies that are derived from mouse hybridomas, for example monoclonal antibodies. In one embodiment, the present invention provides full length anti-IL-1 B antibodies. In another embodiment, the present invention provides anti-IL-1 B optimized antibodies, for example optimized monoclonal anti-IL-1 B antibodies, for example full length optimized monoclonal anti-IL-1 B antibodies. In a further embodiment, the present invention provides an anti-IL-1 B antibody with a reduced number of or with no deamidation and/or deglycosylation sites, for example in the CDRs.
  • the present invention provides an anti-IL-1 B antibody, which has a reduced potential for immunogenicity. In a further embodiment, the present invention provides an anti-IL-1 B antibody, which has a reduced potential for nonspecific binding.
  • Further embodiments encompass DNA molecules encoding antibodies of the present invention, expression vectors and host cells comprising such DNA molecules, and methods of making antibodies of the present invention.
  • the present invention further provides therapeutic uses for the antibodies of the present invention, in particular against respiratory diseases.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:
  • a) a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 (CDR1-L); the amino acid sequence of SEQ ID NO:2 (CDR2-L); and the amino acid sequence of SEQ ID NO:3 (CDR3-L); and b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13 or 1 6 (CDR1-H); the amino acid sequence of SEQ ID NO:14 (CDR2-H); and the amino acid sequence of SEQ ID NO:15 or 17 (CDR3-H); or
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 (CDR1-L); the amino acid sequence of SEQ ID NO:5, 7, 8, 9 or 10 (CDR2-L); and the amino acid sequence of SEQ ID NO:6, 1 1 or 12 (CDR3-L); and d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18 (CDR1-H); the amino acid sequence of SEQ ID NO:19 (CDR2-H); and the amino acid sequence of SEQ ID NO:20 (CDR3-H).
  • the antibody or antigen-binding fragment thereof comprises: a) a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 (CDR1-L); the amino acid sequence of SEQ ID NO:2 (CDR2-L); and the amino acid sequence of SEQ ID NO:3 (CDR3-L); and
  • a heavy chain variable region comprising:
  • the antibody or antigen-binding fragment thereof comprises: a) a light chain variable region comprising:
  • sequence of SEQ ID NO:8 CDR2-L
  • amino acid sequence of SEQ ID NO:1 1 CDR3-L
  • amino acid sequence of SEQ ID NO:4 CDR1-L
  • amino acid sequence of SEQ ID NO:9 CDR2-L
  • amino acid sequence of SEQ ID NO:1 1 CDR3-L
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NO:21 , 25, 26 or 27; and a heavy chain variable region comprising the amino acid sequence any one of SEQ ID NO:23, 34, 35, 36, 37 or 38.
  • the antibody or antigen-binding fragment thereof comprises: i) a light chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23; or
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NO:22, 28, 29, 30, 31 , 32 or 33; and a heavy chain variable region comprising the amino acid sequence any one of SEQ ID NO:24, 39, or 40.
  • the antibody or antigen-binding fragment thereof comprises: i) a light chain variable region comprising the amino acid sequence of SEQ ID NO:22 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24; or
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO:33 and a heavy chain variable region comprising the amino acid sequence one of SEQ ID NO:39.
  • the light chain variable region is linked to a human kappa or lambda light chain constant region and the heavy chain variable region is linked to a human lgG1 , lgG2, lgG3 or lgG4 heavy chain constant region.
  • the antibody is a monoclonal antibody.
  • the present invention provides an antibody comprising the amino acid sequence of any one of SEQ ID NO:34, 35, 36, 37, 38, 39 or 40 linked to a human lgG1 , lgG2, lgG3, lgG4, IgM, IgA or IgE heavy chain constant region, for example a human lgG1 heavy chain constant region.
  • the present invention further provides an antibody comprising the amino acid sequence of any one of SEQ ID NO:25, 26, 27, 28, 29, 30, 31 , 32 or 33 linked to a human kappa or lambda light chain constant region.
  • the present invention provides an antibody or antigen-binding fragment thereof above for use in medicine, for example for treating or preventing a disease in mammals, in particular humans.
  • the use is the treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation, or cancer.
  • the use is for the treatment of Chronic Obstructive Pulmonary Disease (COPD), asthma, Idiopathic Pulmonary Fibrosis (IPF) or Acute Respiratory Distress Syndrom (ARDS).
  • COPD Chronic Obstructive Pulmonary Disease
  • IPF Idiopathic Pulmonary Fibrosis
  • ARDS Acute Respiratory Distress Syndrom
  • the use is for the treatment of Alzheimer's disease, Parkinson's disease, Cryopyrin associated periodic syndrome (CAPS), schizophrenia or epilepsy.
  • the use is for the treatment of diabetic complications, type 1 diabetes, type 2 diabetes, diabetes disease progression, insulin resistance, atherosclerosis, intermittent claudication, peripheral vascular disease, aneurysm, acute coronary syndrome, heart failure, vascular inflammation, myocardial infarction, Uveitis, Behcet's disease, Xerophthalmia or conjunctivitis.
  • the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment as described above and a pharmaceutically acceptable carrier.
  • the present invention further provides a method for treating an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation or cancer comprising
  • administering to a subject in need thereof, for example a patient, an effective amount of an anti-IL-1 B antibody or antigen-binding fragment thereof above, or a
  • the antibody or antigen-binding fragment is administered by a parenteral route of administration, or is administered intravenously or
  • the antibody or antigen-binding fragment is administered subcutaneously.
  • the disease is Chronic Obstructive Pulmonary Disease (COPD), asthma, Idiopathic Pulmonary Fibrosis (IPF) or Acute Respiratory Distress Syndrom (ARDS).
  • COPD Chronic Obstructive Pulmonary Disease
  • IPF Idiopathic Pulmonary Fibrosis
  • ARDS Acute Respiratory Distress Syndrom
  • the disease is Alzheimer's disease, Parkinson's disease, Cryopyrin associated periodic syndrome (CAPS), schizophrenia or epilepsy .
  • the disease is diabetic complications, type 1 diabetes, type 2 diabetes, diabetes disease progression, insulin resistance, atherosclerosis, intermittent claudication, peripheral vascular disease, aneurysm, acute coronary syndrome, heart failure, vascular inflammation, myocardial infarction, Uveitis, Behcet's disease, Xerophthalmia or conjunctivitis.
  • Further embodiments encompass a DNA molecule encoding an antibody or antigen- binding fragment thereof disclosed above.
  • Further embodiments encompass a DNA molecule encoding a variable light chain region above, a DNA molecule encoding a variable heavy chain region above, a DNA molecule encoding a light chain region above or a DNA molecule encoding a heavy chain region above.
  • the present invention provides an isolated polynucleotide comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:21 , 25, 26, or 27, or encoding a heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:23, 34, 35, 36, 37 or 38.
  • the present invention provides an isolated polynucleotide comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:22, 28, 29, 30, 31 , 32 or 33, or encoding a heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:24, 39 or 40.
  • an expression vector containing a DNA molecule above comprises a DNA molecule encoding the constant heavy chain and/or the constant light chain, respectively, linked to the DNA molecule encoding the variable heavy chain and/or the variable light chain, respectively.
  • a host cell carrying one or more expression vectors above In one embodiment, a host is a mammalian cell.
  • Further embodiments encompass a method for producing an antibody or antigen- binding fragment thereof above comprising transfecting a mammalian host cell with one or more of the vectors above, cultivating the host cell and recovering and purifying the antibody or antigen-binding fragment thereof.
  • Further embodiments encompass a method for producing an antibody or antigen- binding fragment thereof comprising obtaining a mammalian host cell comprising one or more of the vectors above, and cultivating the host cell. In one embodiment, the method further comprises recovering and purifying the antibody or antigen-binding fragment thereof. In one embodiment, the present invention further provides an anti-IL-1 B antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a K D for IL-1 B equal to or of less than 25pM, a K D for IL-1 B equal to or of less than 15pM or a K D for IL-1 B equal to or of less than 10pM.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof that competitively binds to human IL-1 B with an antibody of the present invention. In one embodiment, the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof that competitively binds to human IL-1 B with any one of Antibody A1 to A5 or with any one of Antibody B1 to B6.
  • the anti-IL-1 B antibody is an optimized antibody. In one embodiment, the anti-IL-1 B antibody is an optimized antibody.
  • the anti-IL-1 B antibody is a monoclonal antibody. In one embodiment, the anti-IL-1 B antibody is a full length antibody. In one embodiment, the anti-IL-1 B antibody is an optimized monoclonal antibody, for example a full length optimized monoclonal antibody. In one embodiment the antigen-binding fragment is a Fab, F(ab') 2 , or single chain Fv fragment. In one embodiment, the antigen-binding fragment comprises a light chain variable region and a heavy chain variable region.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof comprising a optimized light chain variable domain comprising the CDRs of SEQ ID NO:21 , 25, 26 or 27 and framework regions having an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO: 21 , 25, 26 or 27 and an optimized heavy chain variable domain comprising the CDRs of SEQ ID NO:23, 34, 35, 36, 37 or 38 and framework regions having an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:23, 34, 35, 36, 37 or 38.
  • the anti-IL-1 B antibody is a optimized monoclonal antibody, for example a full length optimized monoclonal antibody.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof comprising a optimized light chain variable domain comprising the CDRs of SEQ ID NO:22, 28, 29, 30, 31 , 32 or 33 and framework regions having an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:22, 28, 29, 30, 31 , 32 or 33 and a optimized heavy chain variable domain comprising the CDRs of SEQ ID NO:24, 39 or 40 and framework regions having an amino acid sequence at least 90% or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:24, 39 or 40.
  • the anti-IL-1 B antibody is a optimized monoclonal antibody, for example a full length optimized monoclonal antibody.
  • the present invention further provides a method for inhibiting the binding of IL-1 B to the IL-1 B receptor on a mammalian cell comprising administering to the cell an antibody molecule or antigen-binding fragment above, whereby signaling mediated by the IL-1 B receptor is inhibited.
  • an antibody of the present invention inhibits this signaling over a wide range of concentrations of IL-1 B, for example physiological and elevated concentrations of human IL-1 B.
  • the present invention further provides a method for treating a subject having an IL-1 B-associated disorder comprising administering to the subject an antibody or antigen-binding fragment above, which antibody or antigen-binding fragment binds to human IL-1 B.
  • the present invention further provides a method for detecting and/or quantifying IL-1 B levels in a biological sample comprising contacting the sample with an antibody or antigen binding fragment above and detecting binding of the antibody or fragment thereof with IL-1 B.
  • This information can be used to diagnose an IL-1 B-associated disorder.
  • methods are provided for diagnosing an IL-1 B- associated disorder or for determining if a subject has an increased risk of developing an IL-1 B-associated disorder, wherein the method comprises contacting a biological sample from a subject with an antibody or antigen binding fragment above and detecting binding of the antibody or antigen binding fragment to IL-1 B to determine the expression or concentration of IL-1 B.
  • the present invention further provides a method for inhibiting the binding of IL-1 B to the IL-1 B receptor on a cell comprising administering to the cell or cellular environment an antibody or antigen-binding fragment above, whereby signaling mediated by the IL-1 B receptor is inhibited.
  • Figure 1 Schematic description of Biacore chip assay.
  • FIG. 1 Biacore Chip assays for antibodies 36C2 (A) and 47F1 1 (B).
  • Figure 3 Determination of cellular potency against mouse IL-1 B for antibody 36C2 and reference antibodies.
  • Figure 4 Cellular studies performed with increasing concentrations of IL-1 B in the presence of a 100-fold molar excess of anti-IL-1 B antibody or control antibody over IL- 1 B.
  • the present invention provides antibodies that bind to IL-1 B, in particular human IL- 1 B.
  • the present invention also relates to optimized antibodies that recognize IL-1 B.
  • the sequence of these optimized antibodies has been identified based on the sequences of certain mouse antibodies.
  • the mouse antibodies of the present invention were derived from mouse hybridomas.
  • the immunization of the mice can be carried out using different techniques know in the art. Preparation of immunogenic antigens and monoclonal antibody production can be performed using any suitable technique known in the art. Such techniques were used in the present invention.
  • the lead mouse antibodies were selected based on their high affinity to human IL-1 B. Accordingly, in one aspect, the present invention provides an antibody that binds to human IL-1 B with high affinity. Selected mouse antibodies were optimized to result in optimized antibodies. The optimized antibodies of the present invention bind to human IL-1 B with high affinity. Accordingly, in another aspect, the present invention provides a optimized antibody that binds to human IL-1 B with high affinity.
  • the present invention further provides an anti-IL-1 B antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a K D for IL-1 B equal to or of less than 25pM, a K D for IL-1 B equal to or of less than 15pM or a K D for IL-1 B equal to or of less than 10pM.
  • an antibody of the present invention inhibits the activity of human IL-1 B in a cell-based assay, for example as described herein, at an IC50 equal to or of less than 50pM, in particular at an IC50 equal to or of less than 30pM. In one aspect, an antibody of the present invention inhibits the activity of human IL-1 B in a cell-based assay, for example as described herein, at an IC50 equal to or of less than 25pM or at an IC50 equal to or of less than 15pM.
  • an antibody of the present invention inhibits the activity of human IL-1 B in a cell-based assay, for example as described herein, at an IC90 equal to or of less than 300pM, in particular at an IC90 equal to or of less than 200pM. In one aspect, an antibody of the present invention inhibits the activity of human IL-1 B in a cell-based assay, for example as described herein, at an IC90 equal to or of less than 150pM or at an IC90 equal to or of less than 10OpM.
  • an antibody of the present invention is able to inhibit the activity of IL- 1 B across different species, for example human IL-1 B, cynomolgus IL-1 B and/or mouse IL-1 B, despite differences in the amino acid sequences of IL-1 B between these species.
  • an antibody of the present invention binds at high affinity to human IL-1 B, cynomolgus IL-1 B and/or mouse IL-1 B.
  • an antibody of the present invention inhibits the activity of cynomolgus IL-1 B in a cell-based assay, for example as described herein, at an IC50 equal to or of less than 50pM, in particular at an IC50 equal to or of less than 40pM. In one aspect, an antibody of the present invention inhibits the activity of cynomolgus IL-1 B in a cell-based assay, for example as described herein, at an IC50 equal to or of less than 20pM.
  • an antibody of the present invention inhibits the activity of cynomolgus IL-1 B in a cell-based assay, for example as described herein, at an IC90 equal to or of less than 300pM, in particular at an IC90 equal to or of less than 250pM. In one aspect, an antibody of the present invention inhibits the activity of cynomolgus IL-1 B in a cell-based assay, for example as described herein at an IC90 equal to or of less than 200pM.
  • an antibody of the present invention to potently inhibit the activity of cynomolgus IL-1 B facilitates the development of clinical candidates in humans, as the cynomolgus monkey is the preferred non-human primate (NHP) species for toxicology studies.
  • the cynomolgus monkey is an Old World monkey of medium size and requires lower amounts of test agent for dosing than the rhesus monkey or baboon. It has historically been the most common species for toxicology testing, including immunotoxicology and reproductive toxicology of human monoclonal antibodies. This allows to test the safety and to accelerate the clinical development of an antibody of the present invention in humans.
  • an antibody of the present invention is capable of inhibiting the activity of mouse IL-1 B.
  • an optimized monoclonal anti-IL-1 B antibody of the present invention has favorable biophysical properties, for example quality, stability, or solubility.
  • an optimized monoclonal anti-IL-1 B antibody of the present invention has reduced non-specific binding, optimized charge, or reduced lipophilic stretches.
  • the anti-IL-1 B antibody is an optimized antibody.
  • the anti- IL-1 B antibody is a monoclonal antibody.
  • the anti-IL-1 B antibody is a full length antibody.
  • the anti-IL-1 B antibody is an optimized monoclonal antibody, for example a full length optimized monoclonal antibody.
  • An antibody or antigen-binding fragment thereof of the present invention recognizes specific "IL-1 B antigen epitope” or " IL-1 B epitope”.
  • these terms refer to a molecule (e.g., a peptide) or a fragment of a molecule capable of immunoreactivity with an anti-IL-1 B antibody and, for example, include an IL-1 B antigenic determinant recognized by any of the antibodies having a light chain/heavy chain sequence combination of SEQ ID NO:21 /22, 23/24, 25/34, 27/35, 27/36, 26/37, 27/38, 28/40, 29/39, 30/40, 31 /40, 32/40 and 33/39.
  • IL-1 B antigen epitopes can be included in proteins, protein fragments, peptides or the like.
  • the epitopes are most commonly proteins, short oligopeptides, oligopeptide mimics (i.e., organic compounds that mimic antibody binding properties of the IL-1 B antigen), or combinations thereof.
  • the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
  • Peptide or polypeptide epitopes contain for example at least seven amino acids or for example at least nine amino acids or for example between about 15 to about 20 amino acids. Since an antibody can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
  • Epitopes may be determined by various techniques known in the art, such as X-ray crystallography, nuclear magnetic resonance, Hydrogen/Deuterium Exchange Mass Spectrometry (HXMS), site-directed mutagenesis, alanine scanning mutagenesis, and peptide screening methods.
  • HXMS Hydrogen/Deuterium Exchange Mass Spectrometry
  • site-directed mutagenesis site-directed mutagenesis
  • alanine scanning mutagenesis alanine scanning mutagenesis
  • peptide screening methods The generalized structure of antibodies or immunoglobulin is well known to those of skill in the art. These molecules are heterotetrameric glycoproteins, typically of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains and are typically referred to as full length antibodies.
  • Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotetrameric molecule is formed through a covalent disulfide linkage between the two identical heavy chains of the heterodimers.
  • the light and heavy chains are linked together by one disulfide bond, the number of disulfide linkages between the two heavy chains varies by immunoglobulin isotype.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at the amino- terminus a variable domain (V H ), followed by three or four constant domains (Cm , CH2 , CH3 , and CH 4 ), as well as a hinge region between Cm and CH2-
  • Each light chain has two domains, an amino-terminal variable domain (V L ) and a carboxy-terminal constant domain (C L ).
  • the V L domain associates non-covalently with the V H domain, whereas the CL domain is commonly covalently linked to the Cm domain via a disulfide bond.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., 1985, J. Mol. Biol. 186:651 -663).
  • Variable domains are also referred herein as variable regions.
  • variable domains differ between different antibodies i.e., are "hypervariable.” These hypervariable domains contain residues that are directly involved in the binding and specificity of each particular antibody for its specific antigenic determinant. Hypervariability, both in the light chain and the heavy chain variable domains, is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991 , in: Sequences of Proteins of Immunological Interest, 5 th Ed.
  • HVLs also referred herein as CDRs
  • CDRs are structurally defined according to the three-dimensional structure of the variable domain, as described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901 -917.
  • CDR-L1 is positioned at about residues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at about residues 89-97 in the light chain variable domain;
  • CDR-H1 is positioned at about residues 31 -35, CDR-H2 at about residues 50-65, and CDR-H3 at about residues 95- 102 in the heavy chain variable domain.
  • the exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
  • the CDR1 , CDR2, CDR3 of the heavy and light chains therefore define the unique and functional properties specific for a given antibody.
  • the three CDRs within each of the heavy and light chains are separated by framework regions (FR), which contain sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4 The largely ⁇ -sheet configuration of the FRs brings the CDRs within each of the chains into close proximity to each other as well as to the CDRs from the other chain. The resulting conformation contributes to the antigen binding site (see Kabat et al., 1991 , NIH Publ. No. 91 -3242, Vol. I, pages 647-669), although not all CDR residues are necessarily directly involved in antigen binding.
  • FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and/or mediate antibody effector function. Some FR residues are thought to have a significant effect on antigen binding in at least three ways: by noncovalently binding directly to an epitope, by interacting with one or more CDR residues, and by affecting the interface between the heavy and light chains.
  • the constant domains are not directly involved in antigen binding but mediate various Ig effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody dependent cellular phagocytosis (ADCP).
  • the light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of the constant domain.
  • the heavy chains of mammalian immunoglobulins are assigned to one of five major classes, according to the sequence of the constant domains: IgA, IgD, IgE, IgG, and IgM.
  • IgG and IgA are further divided into subclasses (isotypes), e.g., IgG-i , lgG 2 , lgG 3 , lgG 4 , IgA-i , and lgA 2 .
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of the classes of native immunoglobulins are well known.
  • antibody specifically encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments such as variable domains and other portions of antibodies that exhibit a desired biological activity, e.g. IL-1 B binding.
  • monoclonal antibody refers to an antibody that is highly specific, being directed against a single antigenic determinant, an "epitope”.
  • monoclonal is indicative of antibodies directed to the identical epitope and is not to be construed as requiring production of the antibody by any particular method. It should be understood that monoclonal antibodies can be made by any technique or methodology known in the art; including e.g., the hybridoma method (Kohler et al., 1975, Nature 256:495), or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No.
  • monomer refers to a homogenous form of an antibody.
  • monomer means a monomeric antibody having two identical heavy chains and two identical light chains.
  • Chimeric antibodies consist of the heavy and light chain variable regions of an antibody from one species (e.g., a non-human mammal such as a mouse) and the heavy and light chain constant regions of another species (e.g., human) antibody and can be obtained by linking the DNA sequences encoding the variable regions of the antibody from the first species (e.g., mouse) to the DNA sequences for the constant regions of the antibody from the second (e.g. human) species and transforming a host with an expression vector containing the linked sequences to allow it to produce a chimeric antibody.
  • a non-human mammal such as a mouse
  • human constant regions of another species
  • the chimeric antibody also could be one in which one or more regions or domains of the heavy and/or light chain is identical with, homologous to, or a variant of the corresponding sequence in a monoclonal antibody from another immunoglobulin class or isotype, or from a consensus or germline sequence.
  • Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, for example binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 6851 -6855).
  • antibody fragment refers to a portion of a full length anti-IL-1 B antibody, in which a variable region or a functional capability is retained, for example specific IL-1 B epitope binding.
  • antibody fragments include, but are not limited to, a Fab, Fab', F(ab') 2 , Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linear antibody, a single-chain antibody, a minibody, a diabody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.
  • Full length antibodies can be treated with enzymes such as papain or pepsin to generate useful antibody fragments.
  • Papain digestion is used to produces two identical antigen-binding antibody fragments called "Fab” fragments, each with a single antigen- binding site, and a residual "Fc” fragment.
  • the Fab fragment also contains the constant domain of the light chain and the Cm domain of the heavy chain.
  • Pepsin treatment yields a F(ab') 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by the presence of additional residues including one or more cysteines from the antibody hinge region at the C-terminus of the Cm domain.
  • F(ab') 2 antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known.
  • Fv fragment contains a complete antigen-recognition and binding site consisting of a dimer of one heavy and one light chain variable domain in tight, non-covalent association.
  • the three CDRs of each variable domain interact to define an antigen-biding site on the surface of the V H -V L dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a “single-chain Fv” or “scFv” antibody fragment is a single chain Fv variant comprising the V H and V L domains of an antibody where the domains are present in a single polypeptide chain.
  • the single chain Fv is capable of recognizing and binding antigen.
  • the scFv polypeptide may optionally also contain a polypeptide linker positioned between the V H and V L domains in order to facilitate formation of a desired three- dimensional structure for antigen binding by the scFv (see, e.g., Pluckthun, 1 994, In The Pharmacology of monoclonal Antibodies, Vol. 1 1 3, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-31 5).
  • a “diabody” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or V.sub.L-V.sub.H).
  • V.sub.H heavy chain variable domain
  • V.sub.L light chain variable domain
  • Diabodies are described more fully in, e.g., Holliger et al. (1 993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
  • an “optimized antibody” or an “optimized antibody fragment” is a specific type of chimeric antibody which includes an immunoglobulin amino acid sequence variant, or fragment thereof, which is capable of binding to a predetermined antigen and which, comprises one or more FRs having substantially the amino acid sequence of a human immunoglobulin and one or more CDRs having substantially the amino acid sequence of a non-human immunoglobulin.
  • This non-human amino acid sequence often referred to as an "import” sequence is typically taken from an "import” antibody domain, particularly a variable domain.
  • an optimized antibody includes at least the CDRs or HVLs of a non-human antibody or derived from a non-human antibody, inserted between the FRs of a human heavy or light chain variable domain.
  • the present invention describes specific optimized anti-IL-1 B antibodies which contain CDRs derived from the mouse monoclonal antibodies or optimized CDRs shown in Tables 3 and 4 inserted between the FRs of human germline sequence heavy and light chain variable domains. It will be understood that certain mouse FR residues may be important to the function of the optimized antibodies and therefore certain of the human germline sequence heavy and light chain variable domains residues are modified to be the same as those of the corresponding mouse sequence. During this process undesired amino acids may also be removed or changed, for example to avoid deamidation, undesirable charges or lipophilicity or non-specific binding.
  • An “optimized antibody”, an “optimized antibody fragment” or “optimized” may sometimes be referred to as "humanized antibody", "humanized antibody fragment” or
  • an optimized anti-IL-1 B antibody comprises substantially all of at least one, and typically two, variable domains (such as contained, for example, in Fab, Fab', F(ab')2, Fabc, and Fv fragments) in which all, or substantially all, of the CDRs correspond to those of a non-human immunoglobulin, and specifically herein, all of the CDRs are mouse or optimized sequences as detailed in Tables 1 through 4 herein below and all, or substantially all, of the FRs are those of a human immunoglobulin consensus or germline sequence.
  • an optimized anti-IL-1 B antibody also includes at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include one or more of the Cm , hinge, C H 2 , C H 3 , and/or C H4 regions of the heavy chain, as appropriate.
  • An optimized anti-IL-1 B antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG-i , lgG 2 , lgG 3 , lgG 4 , IgA ! and lgA 2 .
  • the constant domain can be a complement fixing constant domain where it is desired that the optimized antibody exhibits cytotoxic activity, and the isotype is typically IgG-i . Where such cytotoxic activity is not desirable, the constant domain may be of another isotype, e.g., lgG 2 .
  • An alternative optimized anti-IL-1 B antibody can comprise sequences from more than one immunoglobulin class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the present invention provides antibodies that are lgG1 antibodies and more particularly, are lgG1 antibodies in which there is a knock-out of effector functions.
  • the FRs and CDRs, or HVLs, of an optimized anti-IL-1 B antibody need not correspond precisely to the parental sequences.
  • one or more residues in the import CDR, or HVL, or the consensus or germline FR sequence may be altered (e.g., mutagenized) by substitution, insertion or deletion such that the resulting amino acid residue is no longer identical to the original residue in the corresponding position in either parental sequence but the antibody nevertheless retains the function of binding to IL-1 B.
  • Such alteration typically will not be extensive and will be conservative alterations.
  • at least 75% of the optimized antibody residues will correspond to those of the parental consensus or germline FR and import CDR sequences, more often at least 90%, and most frequently greater than 95%, or greater than 98% or greater than 99%.
  • V L -V H interface Immunoglobulin residues that affect the interface between heavy and light chain variable regions
  • Certain residues that may be involved in interchain interactions include V L residues 34, 36, 38, 44, 46, 87, 89, 91 , 96, and 98 and V H residues 35, 37, 39, 45, 47, 91 , 93, 95, 100, and 103 (utilizing the numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)).
  • V L residues 34, 36, 38, 44, 46, 87, 89, 91 , 96, and 98 V H residues 35, 37, 39, 45, 47, 91 , 93, 95, 100, and 103
  • V L residues 43 and 85 residues such as V L residues 43 and 85, and V H residues 43 and 60 also may be involved in this interaction. While these residues are indicated for human IgG only, they are applicable across species. Important antibody residues that are reasonably expected to be involved in interchain interactions are selected for substitution into the consensus sequence.
  • Consensus sequence and “consensus antibody” refer to an amino acid sequence which comprises the most frequently occurring amino acid residue at each location in all immunoglobulins of any particular class, isotype, or subunit structure, e.g., a human immunoglobulin variable domain.
  • the consensus sequence may be based on immunoglobulins of a particular species or of many species.
  • a "consensus” sequence, structure, or antibody is understood to encompass a consensus human sequence as described in certain embodiments, and to refer to an amino acid sequence which comprises the most frequently occurring amino acid residues at each location in all human immunoglobulins of any particular class, isotype, or subunit structure.
  • the consensus sequence contains an amino acid sequence having at each position an amino acid that is present in one or more known immunoglobulins, but which may not exactly duplicate the entire amino acid sequence of any single immunoglobulin.
  • the variable region consensus sequence is not obtained from any naturally produced antibody or immunoglobulin. Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., and variants thereof.
  • the FRs of heavy and light chain consensus sequences, and variants thereof provide useful sequences for the preparation of optimized anti-IL-1 B antibodies. See, for example, U.S. Pat. Nos. 6,037,454 and 6,054,297.
  • Germline antibody sequences for the light chain of the antibody come from conserved human germline kappa or lambda v-genes and j-genes.
  • the heavy chain sequences come from germline v-, d- and j-genes (LeFranc, M-P, and LeFranc, G, "The Immunoglobulin Facts Book” Academic Press, 2001 ).
  • variant each refers to an optimized anti-IL-1 B antibody having at least a light chain variable murine CDR from any of the sequences as shown in Table 1 or a heavy chain murine CDR sequence derived from the murine monoclonal antibody as shown in Table 2.
  • variants include those having one or more amino acid changes in one or both light chain or heavy chain variable domains, provided that the amino acid change does not substantially impair binding of the antibody to IL-1 B.
  • Exemplary optimized antibodies produced herein include those designated as Antibody A1 , Antibody A2, Antibody A3, Antibody A4, Antibody A5, Antibody B1 , Antibody B2, Antibody B3, Antibody B4, Antibody B5 and Antibody B6.
  • an “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of the antibody's natural environment are those materials that may interfere with diagnostic or therapeutic uses of the antibody, and can be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect, the antibody will be purified to at least greater than 95% isolation by weight of antibody.
  • An isolated antibody includes an antibody in situ within recombinant cells in which it is produced, since at least one component of the antibody's natural environment will not be present. Ordinarily however, an isolated antibody will be prepared by at least one purification step in which the recombinant cellular material is removed.
  • antibody performance refers to factors that contribute to antibody recognition of antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding, and can influence physical factors such as initial rate of antibody binding to antigen (k a ), dissociation constant of the antibody from antigen (k d ), affinity constant of the antibody for the antigen (Kd), non-specific binding, conformation of the antibody, protein stability, and half life of the antibody.
  • epitope tagged when used herein, refers to an anti-IL-1 B antibody fused to an "epitope tag".
  • An "epitope tag” is a polypeptide having a sufficient number of amino acids to provide an epitope for antibody production, yet is designed such that it does not interfere with the desired activity of the optimized anti-IL-1 B antibody.
  • the epitope tag is usually sufficiently unique such that an antibody raised against the epitope tag does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally contain at least 6 amino acid residues and usually contain about 8 to 50 amino acid residues, or about 9 to 30 residues.
  • epitope tags and the antibody that binds the epitope include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., 1988 Mol. Cell. Biol. 8: 2159-21 65; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., 1985, Mol. Cell. Biol. 5(12):3610- 3616; and Herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. 1990, Protein Engineering 3(6): 547-553).
  • the epitope tag is a "salvage receptor binding epitope".
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (such as IgG-i , lgG 2 , lgG 3 , or lgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the antibodies of the present invention may be conjugated to a cytotoxic agent.
  • a cytotoxic agent This is any substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (such as I 131 , I 125 , Y 90 , and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer. There are numerous examples of chemotherapeutic agents that could be conjugated with the therapeutic antibodies of the present invention.
  • the antibodies also may be conjugated to prodrugs.
  • a "prodrug” is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active form. See, for example, Wilman, 1986, “Prodrugs in Cancer Chemotherapy", In Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor and Stella et al., 1985, “Prodrugs: A Chemical Approach to Targeted Drug Delivery, In: “Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press.
  • Useful prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs peptide- containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ - lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, and optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouridine prodrugs that can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be derivatized into a prodrug form include, but are not limited to, those chemotherapeutic agents described above.
  • the antibodies of the invention also may be conjugated to a label, either a label alone or a label and an additional second agent (prodrug, chemotherapeutic agent and the like).
  • a label as distinguished from the other second agents refers to an agent that is a detectable compound or composition and it may be conjugated directly or indirectly to an optimized antibody of the present invention.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • Labeled optimized anti-IL-1 B antibody can be prepared and used in various applications including in vitro and in vivo diagnostics.
  • the antibodies of the present invention may be formulated as part of a liposomal preparation in order to affect delivery thereof in vivo.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids, and/or surfactant. Liposomes are useful for delivery to a mammal of a compound or formulation, such as an optimized anti-IL-1 B antibody disclosed herein, optionally, coupled to or in combination with one or more pharmaceutically active agents and/or labels.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Certain aspects of the present invention relate to isolated nucleic acids that encode one or more domains of the antibodies of the present invention, for example optimized antibodies of the present invention.
  • An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is distinguished from the nucleic acid molecule as it exists in natural cells.
  • one or more domains of the optimized antibodies will be expressed in a recombinant form.
  • Such recombinant expression may employ one or more control sequences, i.e., polynucleotide sequences necessary for expression of an operably linked coding sequence in a particular host organism.
  • the control sequences suitable for use in prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences.
  • Eukaryotic control sequences include, but are not limited to, promoters, polyadenylation signals, and enhancers. These control sequences can be utilized for expression and production of optimized anti-IL- 1 B antibody in prokaryotic and eukaryotic host cells.
  • a nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame.
  • enhancers are optionally contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used.
  • the expressions "cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include the progeny thereof.
  • “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like.
  • the mammal is human.
  • a “disorder”, as used herein, is any condition that would benefit from treatment with a optimized anti-IL-1 B antibody described herein. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.
  • disorders to be treated herein include inflammatory, angiogenic, autoimmune and immunologic disorders, respiratory disorders, central nervous system disorders, eye disorders, cardiovascular disorders, cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • IL-1 B-associated disorder or "IL-1 B -associated disease” refers to a condition in which IL-1 B activity contributes to the disease, for example where active IL-1 B or its endogenous antagonists are present at abnormal levels.
  • An IL-1 B-associated disorder includes diseases and disorders of the immune system, such as autoimmune disorders and inflammatory disorders.
  • An IL-1 B-associated disorder also includes respiratory diseases.
  • Such conditions include, but are not limited to Cryopyrin associated periodic syndrome (CAPS), Chronic Obstructive Pulmonary Disease (COPD). Asthma, Idiopathic Pulmonary Fibrosis (IPF) or Acute Respiratory Distress Syndrome (ARDS), septic shock, Alzheimer's disease.
  • Cryopyrin associated periodic syndrome Cryopyrin associated periodic syndrome
  • COPD Chronic Obstructive Pulmonary Disease
  • Asthma Asthma
  • Idiopathic Pulmonary Fibrosis Idiopathic Pulmonary Fibrosis
  • ARDS Acute Respiratory Distress Syndrome
  • septic shock Alzheimer's disease.
  • Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, multiple sclerosis, psoriasis, psoriatic arthritis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), pulmonary inflammation, asthma, idiopathic thrombocytopenic purara (ITP) and ankylosing spondylitis.
  • intravenous infusion refers to introduction of an agent into the vein of an animal or human patient over a period of time greater than approximately 15 minutes, generally between approximately 30 to 90 minutes.
  • intravenous bolus or “intravenous push” refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, generally 5 minutes or less.
  • subcutaneous administration refers to introduction of an agent under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle. Pinching or drawing the skin up and away from underlying tissue may create the pocket.
  • subcutaneous infusion refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less.
  • the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen.
  • subcutaneous bolus refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is less than approximately 15 minutes; in another aspect, less than 5 minutes, and in still another aspect, less than 60 seconds. In yet even another aspect, administration is within a pocket between the skin and underlying tissue, where the pocket may be created by pinching or drawing the skin up and away from underlying tissue.
  • therapeutically effective amount is used to refer to an amount of an active agent that relieves or ameliorates one or more of the symptoms of the disorder being treated. In another aspect, the therapeutically effective amount refers to a target serum concentration that has been shown to be effective in, for example, slowing disease progression. Efficacy can be measured in conventional ways, depending on the condition to be treated.
  • treatment and “therapy” and the like, as used herein, are meant to include therapeutic as well as prophylactic, or suppressive measures for a disease or disorder leading to any clinically desirable or beneficial effect, including but not limited to alleviation or relief of one or more symptoms, regression, slowing or cessation of progression of the disease or disorder.
  • treatment includes the administration of an agent prior to or following the onset of a symptom of a disease or disorder thereby preventing or removing one or more signs of the disease or disorder.
  • the term includes the administration of an agent after clinical manifestation of the disease to combat the symptoms of the disease.
  • compositions of the invention either alone or in combination with another therapeutic agent alleviate or ameliorate at least one symptom of a disorder being treated as compared to that symptom in the absence of use of the optimized anti-IL-1 B antibody composition, the result should be considered an effective treatment of the underlying disorder regardless of whether all the symptoms of the disorder are alleviated or not.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • anti-IL-1 B antibodies in particular optimized anti-IL-1 B antibodies, and compositions and articles of manufacture comprising one or more anti-IL-1 B antibody, in particular one or more optimized anti- IL-1 B antibody of the present invention.
  • binding agents that include an antigen-binding fragment of an anti-IL-1 B antibody, in particular an optimized anti- IL-1 B antibody.
  • the optimized anti-IL-1 B antibodies and binding agents can inhibit or reduce the production of inflammatory cytokines, for example IL-6, from a variety of cell types including, but not exclusively fibroblasts, epithelial cells, endothelial cells, monocytes, macrophages, neutrophils, which together contribute to diseases where IL-1 B mediated inflammation is detrimental.
  • the optimized anti-IL-1 B antibodies and binding agents can thus be used in the treatment of a variety of diseases or disorders.
  • An optimized anti-IL-1 B antibody and an IL-1 B binding agent each includes at least a portion that specifically recognizes an IL-1 B epitope (i.e., an antigen-binding fragment).
  • Selected mouse antibodies have the following light chain variable regions and heavy chain variable regions as shown in Table 1 and 2:
  • Human framework sequences were selected for each of the mouse leads based on the framework homology, CDR structure, conserved canonical residues, conserved interface packing residues and other parameters.
  • mice light chain and heavy chain CDRs of the various mouse antibodies are shown in Table 3 and Table 4, respectively.
  • Tables 3 and 4 also show light chain and heavy chain CDRs derived from mouse antibodies 36C2 and 47F1 1 through the optimization process.
  • the CDRs listed above in Tables 3 and 4 are defined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273,927-948). Fabs that showed better or equal binding as compared to the chimeric parent Fab were selected for conversion to IgG. 36C2 and 47F1 1 were converted to an IgGI KO format. IgGI KO (knock-out of effector functions) has two mutations in the Fc region, Leu234Ala and Leu235Ala, which reduce effector function such as FcyR and complement binding. The IgG format is described in the literature (see for example Hezareh et al. (2001 ) Journal of Virology 75: 121 61 -121 68). Example 5 describes the sequence-optimization process in further detail. The sequence optimization resulted in optimized antibody sequences. A representative number of optimized light chain and heavy chain variable regions derived from mouse antibodies 36C2 and 47F1 1 are provided and shown in Tables 5 and 6.
  • NKELPWTFGQGTKLEIK (SEQ ID NO : 26 )
  • Table 7 shows examples of heavy and light chain variable regions pairing to prod Antibodies A1 to A5 and B1 to B6.
  • an optimized anti-IL-1 B antibody of the present invention has one or more of the properties below.
  • an antibody of the present invention has two or more, three or more, four or more, five or more, six or more, or seven or more of the properties below.
  • an optimized anti-IL-1 B antibody of the present invention has one or more of the properties below.
  • an antibody of the present invention has two or more, three or more, four or more, five or more, six or more, or all the properties below.
  • IL-18 or IL1 RA concentrations of IL-18 or IL1 RA. • Inhibits human and cynomolgus IL-1 B induced SEAP (IL-1 B stimulated NFkB, AP1 activity) from the HEK-blue reporter cell line with IC 50 values ⁇ 25 pM, for example ⁇ 20 pM.
  • an optimized anti-IL-1 B antibody of the present invention has one or more the properties below.
  • an antibody of the present invention has two or more, three or more, four or more, five or more, six or more, or seven or more of the properties below.
  • ⁇ Inhibits IL6 production from human PBMCs that have been primed with E. coli LPS and subsequently stimulated with ATP and IL12.
  • the optimized antibody displays inhibiting activity, whereby it decreases the binding of IL-1 B to IL-1 receptor by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95%.
  • the ability of an antibody to inhibit binding of IL-1 B to the IL-1 receptor can be measured using competitive binding assays known in the art.
  • the inhibiting activity of an antibody can be measured by assessing the biological effects of IL-1 B, such as the production of cytokines such as IL6 or IL8 to determine if signaling mediated by the IL-1 receptor is inhibited.
  • an antibody of the present invention is capable of inhibiting the activity of human IL-1 B over a wide range of concentrations of IL-1 B, including high concentrations of IL-1 B.
  • high concentrations of antibody and ligand complex may be reached in human patients following the dosing of an anti-IL-1 B antibody of the present invention.
  • the present invention provides a humanized anti-IL-1 B antibody having favorable biophysical properties.
  • an optimized anti-IL-1 B antibody of the present invention is present in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer.
  • an optimized anti-IL-1 B antibody of the present invention remains in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof that competitively binds to human IL-1 B with an antibody of the present invention, for example Antibody A1 , Antibody A2, Antibody A3, Antibody A4, Antibody A5, Antibody B1 , Antibody B2, Antibody B3, Antibody B4, Antibody B5 or Antibody B6 described herein.
  • an antibody or antigen- binding fragment to competitively bind to IL-1 B can be measured using competitive binding assays known in the art.
  • the optimized anti-IL-1 B antibodies optionally include specific amino acid substitutions in the consensus or germline framework regions.
  • the specific substitution of amino acid residues in these framework positions can improve various aspects of antibody performance including binding affinity and/or stability, over that demonstrated in optimized antibodies formed by "direct swap" of CDRs or HVLs into the human germline framework regions.
  • the present invention describes monoclonal antibodies with a light chain variable region having the amino acid sequence set forth in of SEQ ID NO: 21 or 22.
  • the present invention describes other monoclonal antibodies with a heavy chain variable region having the amino acid sequence set forth in of SEQ ID NO: 23 or 24 (see Tables 1 and 2 above).
  • the CDR sequence of these mouse antibodies are shown in Tables 3 and 4. Placing such CDRs into FRs of the human consensus heavy and light chain variable domains yields useful optimized antibodies of the present invention.
  • the present invention provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable regions of SEQ ID NO:21 /22 or 23/24. Such variable regions can be combined with human constant regions.
  • the present invention describes optimized antibodies with light chain variable region sequences having the amino acid sequence set forth in any one of SEQ ID NO:25-27 or 28-33.
  • the present invention describes optimized antibodies with heavy chain variable region sequences having the amino acid sequence set forth in any one of SEQ ID NO:34-38 or 39-40 (see Tables 5 and 6 above). The CDR sequences of these antibodies are shown in Tables 3 and 4.
  • the present invention provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable regions of SEQ ID NO: 25/34, 27/35, 27/36, 26/37, 27/38, 28/40, 29/39, 30/40, 31 /40, 32/40 or 33/39.
  • Such variable regions can be combined with human constant regions.
  • the present invention relates to an anti-IL-1 B antibody or antigen-binding fragment thereof comprising a optimized light chain variable domain comprising the CDRs of SEQ ID NO:21 , 25, 26 or 27 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO: 21 , 25, 26 or 27 and a optimized heavy chain variable domain comprising the CDRs of SEQ ID NO:23, 34, 35, 36, 37 or 38 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:23, 34, 35, 36, 37 or 38.
  • the anti-IL-1 B antibody is a optimized monoclonal antibody, for example a full length optimized monoclonal antibody.
  • the optimized anti-IL-1 B antibodies disclosed herein comprise at least a heavy or a light chain variable domain comprising the CDRs or HVLs of the murine monoclonal antibodies or optimized antibodies as shown in Tables 1 through 6 above and the FRs of the human germline heavy and light chain variable domains.
  • the present invention provides an anti-IL-1 B antibody or antigen-binding fragment thereof comprising a light chain CDR1 (L-CDR1 ) sequence of SEQ ID NO:1 or 4; a light chain CDR2 (L-CDR2) sequence of SEQ ID NO:2, 5, 7, 8, 9 or 10; a light chain CDR3 (L-CDR3) sequence of SEQ ID NO:3, 6, 1 1 or 12; a heavy chain CDR1 (H-CDR1 ) sequence of SEQ ID NO:13, 16 or 18; a heavy chain CDR2 (H-CDR2) sequence of SEQ ID NO:14 or 19; and a heavy chain CDR3 (H-CDR3) sequence of SEQ ID NO:15, 17 or 20.
  • the anti-IL-1 B antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1 listed above, a L-CDR2 listed above and a L-CDR3 listed above, and a heavy chain variable region comprising a H-CDR1 listed above, a H-CDR2 listed above and a H-CDR3 listed above.
  • the present invention provides an anti-IL-1 B antibody or antigen- binding fragment thereof comprising:
  • the anti-IL-1 B antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1 , L-CDR2 and L-CDR3 combination listed above, and a heavy chain variable region comprising a H-CDR1 , H-CDR2 and H- CDR3 combination listed above.
  • chimeric antibodies with switched CDR regions i.e., for example switching one or two CDRs of one of the mouse antibodies or optimized antibody derived therefrom with the analogous CDR from another mouse antibody or optimized antibody derived therefrom
  • the optimized anti-IL-1 B antibody is an antibody fragment.
  • fragments have been generally discussed above and there are techniques that have been developed for the production of antibody fragments. Fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-1 17; and Brennan et al., 1985, Science 229:81 ). Alternatively, the fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (see, e.g., Carter et al., 1992, Bio/Technology 10:1 63-167).
  • F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the present invention provides antibody fragments comprising the CDRs described herein, in particular one of the combinations of L-CDR1 , L-CDR2, L-CDR3, H-CDR1 , H-CDR2 and H-CDR3 described herein.
  • the present invention provides antibody fragments comprising the variable regions described herein, for example one of the combinations of light chain variable regions and heavy chain variable regions described herein.
  • the antibody or antibody fragment includes a constant region that mediates effector function.
  • the constant region can provide antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) responses.
  • the effector domain(s) can be, for example, an Fc region of an Ig molecule.
  • the effector domain of an antibody can be from any suitable vertebrate animal species and isotypes.
  • the isotypes from different animal species differ in the abilities to mediate effector functions.
  • the ability of human immunoglobulin to mediate CDC and ADCC/ADCP is generally in the order of and respectively.
  • Murine immunoglobulins mediate CDC and ADCC/ADCP generally in the order of murine and lgG2b>lgG 2 a>lgGi »lgG 3 , respectively.
  • murine lgG 2a mediates ADCC while both murine lgG 2a and IgM mediate CDC.
  • the optimized anti-IL-1 B antibodies and agents can include modifications of the optimized anti-IL-1 B antibody or antigen-binding fragment thereof. For example, it may be desirable to modify the antibody with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer.
  • One such modification is the introduction of cysteine residue(s) into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, for example, Caron et al., 1992, J. Exp Med. 176:1 191 -1 195; and Shopes, 1992, J. Immunol. 148:2918- 2922.
  • Homodimeric antibodies having enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., 1993, Cancer Research 53: 2560-2565.
  • an antibody can be engineered to contain dual Fc regions, enhancing complement lysis and ADCC capabilities of the antibody. See Stevenson et al., 1989, Anti-Cancer Drug Design 3: 219-230.
  • Antibodies with improved ability to support ADCC have been generated by modifying the glycosylation pattern of their Fc region. This is possible since antibody glycosylation at the asparagine residue, N297, in the CH2 domain is involved in the interaction between IgG and Fey receptors prerequisite to ADCC.
  • Host cell lines have been engineered to express antibodies with altered glycosylation, such as increased bisecting N- acetylglucosamine or reduced fucose. Fucose reduction provides greater enhancement to ADCC activity than does increasing the presence of bisecting N-acetylglucosamine.
  • enhancement of ADCC by low fucose antibodies is independent of the FcYRIIIa V/F polymorphism.
  • Modifying the amino acid sequence of the Fc region of antibodies is an alternative to glycosylation engineering to enhance ADCC.
  • the binding site on human IgGi for Fey receptors has been determined by extensive mutational analysis. This led to the generation of optimized IgGi antibodies with Fc mutations that increase the binding affinity for FcyRllla and enhance ADCC in vitro. Additionally, Fc variants have been obtained with many different permutations of binding properties, e.g., improved binding to specific FcyR receptors with unchanged or diminished binding to other FcyR
  • immunoconjugates comprising the optimized antibody or fragments thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used to form useful immunoconjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, the tricothecenes, and the like.
  • a variety of radionuclides are available for the production of radioconjugated optimized anti-IL-1 B antibodies. Examples include 212 Bi, 131 1,
  • Conjugates of the optimized anti-IL-1 B antibody and cytotoxic or chemotherapeutic agent can be made by known methods, using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4- dinitrobenz
  • a ricin immunotoxin can be prepared as described in Vitetta et al., 1987, Science 238:1098.
  • Carbon-14-labeled 1 -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
  • Conjugates also can be formed with a cleavable linker.
  • the optimized anti-IL-1 B antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545.
  • Liposomes having enhanced circulation time are disclosed, for example, in U.S. Pat. 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.
  • Fab' fragments of an antibody disclosed herein can be conjugated to the liposomes as described in Martin et al., 1982, J. Biol. Chem. 257:286-288 via a disulfide interchange reaction.
  • a chemotherapeutic agent such as doxorubicin is optionally contained within the liposome. See, e.g., Gabizon et al., 1989, J. National Cancer Inst. 81 (19):1484.
  • the antibodies described and disclosed herein can also be used in ADEPT (Antibody- Directed Enzyme Prodrug Therapy) procedures by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent), to an active anti-cancer drug.
  • ADEPT Antibody- Directed Enzyme Prodrug Therapy
  • the enzyme component of the immunoconjugate useful for ADEPT is an enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Specific enzymes that are useful in ADEPT include, but are not limited to, alkaline phosphatase for converting phosphate-containing prodrugs into free drugs; arylsulfatase for converting sulfate-containing prodrugs into free drugs; cytosine deaminase for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase for converting glycosylated prodrugs into free drugs; ⁇ -lactamase for converting drugs
  • antibodies having enzymatic activity can be used to convert the prodrugs into free active drugs (see, for example, Massey, 1987, Nature 328: 457-458).
  • Antibody-abzyme conjugates can be prepared by known methods for delivery of the abzyme to a tumor cell population, for example, by covalently binding the enzyme to the optimized anti-IL-1 B antibody/heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody disclosed herein linked to at least a functionally active portion of an enzyme as described above can be constructed using recombinant DNA techniques (see, e.g., Neuberger et al., 1984, Nature 312:604-608).
  • an optimized anti-IL-1 B antibody fragment rather than an intact antibody, to increase tissue penetration, for example. It may be desirable to modify the antibody fragment in order to increase its serum half life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment.
  • the appropriate region of the antibody fragment can be altered (e.g., mutated), or the epitope can be incorporated into a peptide tag that is then fused to the antibody fragment at either end or in the middle, for example, by DNA or peptide synthesis. See, e.g., WO 96/32478.
  • covalent modifications of the optimized anti-IL-1 B antibody are also included.
  • Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl, or threonyl residues.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. Such modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable.
  • Other types of covalent modifications of the antibody can be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the amino- or carboxy-terminal residues.
  • Another type of useful covalent modification comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in one or more of U.S. Pat. No. 4,640,835, U.S. Pat. No. 4,496,689, U.S. Pat. No. 4,301 ,144, U.S. Pat. No. 4,670,417, U.S. Pat. No. 4,791 ,192 and U.S. Pat. No. 4,179,337.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • Amino acid sequence variants of the anti-IL-1 B antibody can be prepared by introducing appropriate nucleotide changes into the anti-IL-1 B 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 the anti-IL-1 B antibodies of the examples herein. Any combination of deletions, insertions, and substitutions 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 optimized or variant anti-IL-1 B antibody, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the anti-IL-1 B antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (Science, 244:1081 -1085 (1989)).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (typically alanine) to affect the interaction of the amino acids with IL-1 B 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.
  • alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-IL-1 B antibody variants are screened for the desired activity.
  • 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 anti-IL-1 B antibody fused to an epitope tag.
  • Other insertional variants of the anti-IL-1 B antibody molecule include a fusion to the N- or C- terminus of the anti-IL-1 B antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-IL-1 B antibody molecule removed and a different residue inserted in its place.
  • substitutional mutagenesis sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 5 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions", or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • the biological properties of the antibody can be 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:
  • hydrophobic norleucine, met, ala, val, leu, ile;
  • neutral hydrophilic cys, ser, thr;
  • cysteine residue not involved in maintaining the proper conformation of the optimized or variant anti-IL-1 B antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule, prevent aberrant crosslinking, or provide for established points of conjugation to a cytotoxic or cytostatic compound.
  • 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).
  • a type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., an optimized or human antibody).
  • a parent antibody e.g., an optimized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity).
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody.
  • altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • glycosylations sites it may be desirable to modify the antibodies of the invention to add glycosylations sites.
  • 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.
  • the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
  • O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • a given protein e.g., an antibody
  • the amino acid sequence of the protein is engineered to contain 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).
  • Nucleic acid molecules encoding amino acid sequence variants of the anti-IL-1 B antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site- directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-IL-1 B antibody.
  • isolated polynucleotides that comprise a sequence encoding an optimized anti-IL-1 B antibody, vectors, and host cells comprising the polynucleotides, and recombinant techniques for production of the optimized antibody.
  • the isolated polynucleotides can encode any desired form of the anti-IL-1 B antibody including, for example, full length monoclonal antibodies, Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • Some embodiments include isolated polynucleotides comprising sequences that encode the light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO: SEQ ID NO: 21 , 25, 26, or 27. Some embodiments include isolated polynucleotides comprising sequences that encode the light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO: SEQ ID NO: 22, 28, 29, 30, 31 , 32 or 33.
  • Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 23, 34, 35, 36, 37 or 38. Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 24, 39 or 40.
  • the polynucleotide(s) that comprise a sequence encoding an optimized anti-IL-1 B antibody or a fragment or chain thereof can be fused to one or more regulatory or control sequence, as known in the art, and can be contained in suitable expression vectors or host cell as known in the art.
  • Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling the production of intact antibodies.
  • polynucleotides, or portions thereof can be fused together, providing a template for production of a single chain antibody.
  • a polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning amplification of the DNA
  • vectors for expressing the recombinant antibody are available.
  • the vector components 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, an enhancer element, a promoter, and a transcription termination sequence.
  • the optimized anti-IL-1 B antibodies can also be produced as fusion polypeptides, in which the antibody is fused with a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence can be substituted by a prokaryotic signal sequence.
  • the signal sequence can be, for example, alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leaders, and the like.
  • yeast secretion the native signal sequence can be substituted, for example, with a leader sequence obtained from yeast invertase alpha-factor (including Saccharomyces and Kluyveromyces a-factor leaders), acid phosphatase, C.
  • albicans glucoamylase or the signal described in WO90/13646.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, can be used.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the optimized anti-IL-1 B antibody.
  • Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2- ⁇ . plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification of expression.
  • Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, or alternatively, are complement auxotrophic deficiencies, or in other alternatives supply specific nutrients that are not present in complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.
  • Common selectable markers for mammalian cells are those that enable the identification of cells competent to take up a nucleic acid encoding an optimized anti-IL-1 B antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-l and -II (such as primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • metallothionein-l and -II such as primate metallothionein genes
  • adenosine deaminase ornithine decarboxylase
  • Cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a
  • host cells can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.
  • selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.
  • the TRP1 gene present in the yeast plasmid YRp7 can be used as a selectable marker.
  • the TRP1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12).
  • the presence of the trpi lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented by known plasmids bearing the LEU2 gene.
  • vectors derived from the 1 .6 pm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts.
  • an expression system for large- scale production of recombinant calf chymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology 8:135).
  • Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed (Fleer et al., 1991 , Bio/Technology 9:968-975).
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molecule encoding an anti-IL-1 B antibody or polypeptide chain thereof.
  • Promoters suitable for use with prokaryotic hosts include phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the optimized anti-IL-1 B antibody.
  • S.D. Shine-Dalgarno
  • eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Inducible promoters have the additional advantage of transcription controlled by growth conditions.
  • yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization include yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Optimized anti-IL-1 B antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, or from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindi 11 E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601 ,978.
  • Rous sarcoma virus long terminal repeat can be used as the promoter.
  • enhancer sequence Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to increase the transcription of a DNA encoding an optimized anti-IL-1 B antibody by higher eukaryotes.
  • enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, a-fetoprotein, and insulin).
  • an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the optimized anti-IL-1 B antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
  • telomere sequences transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-IL-1 B antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/1 1026 and the expression vector disclosed therein.
  • optimized anti-IL-1 B antibodies can be expressed using the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809; the disclosure of which is incorporated by reference herein.)
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli 294 ATCC 31 ,446
  • E. coli B E. coli X1776
  • E. coli W31 10 ATCC 27,325
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for optimized anti-IL-1 B antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 1 6,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastors EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated optimized anti-IL-1 B antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells, including, e.g., numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm).
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • expression of optimized anti-IL-1 B is carried out in vertebrate cells.
  • the propagation of vertebrate cells in culture has become routine procedure and techniques are widely available.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1 651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., 1977, J. Gen Virol.
  • monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51 ), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for optimized anti-IL-1 B antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce an optimized anti-IL-1 B antibody described herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1 640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) are suitable for culturing the host cells.
  • Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, U.S. Pat. No. 5,122,469, WO 90/103430, and WO 87/00195 may be used as culture media for the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source.
  • growth factors such as insulin, transferrin, or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium, and phosphate
  • buffers such as HEPES
  • nucleotides such as adenosine and thymidine
  • antibiotics such as gentamicin
  • trace elements defined as inorganic compounds usually present at final concentrations in the micromolar range
  • glucose or an equivalent energy source glucose or an equivalent energy source.
  • Other supplements may also be included at appropriate concentrations that would be
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the cells may be disrupted to release protein as a first step. Particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 1 0:1 63-1 67 describes a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes.
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • a variety of methods can be used to isolate the antibody from the host cell.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique.
  • affinity chromatography is a typical purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human gammal , gamma2, or gamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1 -13).
  • Protein G is recommended for all mouse isotypes and for human gamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575).
  • a matrix to which an affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a Cm domain
  • the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, N.J.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, typically performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • nucleic acids that hybridize under low, moderate, and high stringency conditions, as defined herein, to all or a portion (e.g., the portion encoding the variable region) of the nucleotide sequence represented by isolated polynucleotide sequence(s) that encode an antibody or antibody fragment of the present invention.
  • the hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or 50) nucleotides in length.
  • hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding an anti-IL-1 B polypeptide (e.g., a heavy chain or light chain variable region), or its complement.
  • Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, e.g., a PCR primer, or a diagnostic probe.
  • the determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is incorporated into the N BLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl.
  • protein sequence alignment may be carried out using the CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.
  • the antibodies described herein are useful as affinity purification agents.
  • the antibodies are immobilized on a solid phase such a Protein A resin, using methods well known in the art.
  • the immobilized antibody is contacted with a sample containing the IL-1 B protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the IL-1 B protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the IL-1 B protein from the antibody.
  • Anti-IL-1 B antibodies for example optimized anti-IL-1 B antibodies, are also useful in diagnostic assays to detect and/or quantify IL-1 B protein, for example, detecting IL-1 B expression in specific cells, tissues, or serum.
  • the anti-IL-1 B antibodies can be used diagnostically to, for example, monitor the development or progression of a disease as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the anti-IL-1 B antibody.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • the anti-IL-1 B antibodies can be used in methods for diagnosing an IL-1 B-associated disorder (e.g., a disorder characterized by abnormal concentrations of IL-1 B) or to determine if a subject has an increased risk of developing an IL-1 B-associated disorder.
  • Such methods include contacting a biological sample from a subject with an IL-1 B antibody and detecting binding of the antibody to IL-1 B.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-1 B. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
  • the method can further comprise comparing the level of IL-1 B in a patient sample to a control sample (e.g., a subject that does not have an IL-1 B- associated disorder) to determine if the patient has an IL-1 B-associated disorder or is at risk of developing an IL-1 B-associated disorder.
  • a control sample e.g., a subject that does not have an IL-1 B- associated disorder
  • the antibody may be labeled with a detectable moiety.
  • detectable labels including radioisotopes, fluorescent labels, enzyme substrate labels and the like.
  • the label may be indirectly conjugated with the antibody using various known techniques.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody can be conjugated with a small hapten (such as digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten such as digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • radioisotopes labels include 35 S, 14 C, 125 l, 3 H, and 131 1.
  • the antibody can be labeled with the radioisotope, using the techniques described in, for example, Current Protocols in Immunology, Volumes 1 and 2, 1991 , Coligen et al., Ed. Wiley-lnterscience, New York, N.Y., Pubs. Radioactivity can be measured, for example, by scintillation counting.
  • Exemplary fluorescent labels include labels derived from rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody via known techniques, such as those disclosed in Current Protocols in Immunology, for example. Fluorescence can be quantified using a fluorimeter.
  • enzyme-substrate labels known in the art (see, e.g., U.S. Pat. No. 4,275,149 for a review). The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques.
  • alteration may be a color change in a substrate that can be measured spectrophotometrically.
  • the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured, using a chemiluminometer, for example, or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocydic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • glucose oxidase galactose oxidase
  • enzyme-substrate combinations include, for example: Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylene diamine (OPD) or 3,3',5,5'- tetramethyl benzidine hydrochloride (TMB); alkaline phosphatase (AP) with para- Nitrophenyl phosphate as chromogenic substrate; and ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate such as p-nitrophenyl- -D-galactosidase or fluorogenic substrate 4-methylumbelliferyl- -D-galactosidase.
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'- tetramethyl benzidine hydrochloride
  • AP alkaline phosphatase
  • AP
  • the optimized anti-IL-1 B antibody is used unlabeled and detected with a labeled antibody that binds the optimized anti-IL-1 B antibody.
  • the antibodies described herein may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
  • the anti-IL-1 B antibody or antigen binding fragment thereof can be used to inhibit the binding of IL-1 B to the IL-1 receptor.
  • Such methods comprise administering an anti-IL- 1 B antibody or antigen binding fragment thereof to a cell (e.g., a mammalian cell) or cellular environment, whereby signaling mediated by the IL-1 B receptor is inhibited. These methods can be performed in vitro or in vivo.
  • cellular environment is intended the tissue, medium, or extracellular matrix surrounding a cell.
  • the anti-IL-1 B antibody or antigen binding fragment thereof is administered to the cellular environment of a cell in such a manner that the antibody or fragment is capable of binding to IL-1 B molecules outside of and surrounding the cell, therefore, preventing the binding of IL-1 B to its receptor. Diagnostic Kits
  • An anti-IL-1 B antibody can be used in a diagnostic kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit may include substrates and cofactors required by the enzyme such as a substrate precursor that provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (for example a block buffer or lysis buffer), and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
  • an optimized anti-IL-1 B antibody disclosed herein is useful in the treatment of various disorders associated with the expression of IL-1 B as described herein.
  • Methods for treating an IL-1 B associated disorder comprise administering a therapeutically effective amount of an optimized anti-IL-1 B antibody to a subject in need thereof.
  • the optimized anti-IL-1 B antibody or agent is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, intra-ocular, transdermal, topical, orally inhaled and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration (including perfusing or otherwise contacting the graft with the antibody before transplantation).
  • the optimized anti-IL-1 B antibody or agent can be administered, for example, as an infusion or as a bolus.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intra-articular, or subcutaneous administration.
  • the optimized anti-IL-1 B antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the appropriate dosage of antibody will depend on a variety of factors such as the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 20 mg/kg (e.g., 0.1 - 15 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • suppression is used herein in the same context as “amelioration” and “alleviation” to mean a lessening of one or more characteristics of the disease.
  • the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder associated with detrimental IL-1 B activity.
  • the antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of optimized anti-IL-1 B antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
  • the anti-IL-1 B antibodies or agents are useful for treating or preventing an immunological disorder characterized by abnormal activity of IL-1 B or its naturally occurring human antagonists, e.g., by inappropriate activation of immune cells (e.g. monocytes, epithelial cells, endothelial cells, lymphocytes, macrophages, microglia or dendritic cells) and by acting on other cells types (e.g. fibroblasts) whose activation indirectly effects immune cells.
  • Such abnormal concentrations of IL-1 B can be due to, for example, increased IL-1 B protein levels or, for example, by reduced levels of endogenous IL1 B antagonists (e.g., IL1 Ra, soluble IL1 RII).
  • the anti-IL-1 B antibodies or antigen binding fragments thereof also find use in the treatment or prevention of respiratory disorders, metabolic disorders, for example diabetes mellitus, and certain cancers.
  • Treatment or prevention of the immunological disorder, respiratory disorder, metabolic disorder or cancer, according to the methods described herein, is achieved by administering to a subject in need of such treatment or prevention an effective amount of the anti-IL-1 B antibody or agent, whereby the antibody inhibits the activity of IL-1 B or effector molecules induced by IL1 B associated with the disease state.
  • Immunological diseases that are characterized by inappropriate activation of immune cells and that can be treated or prevented by the methods described herein can be classified, for example, by the type(s) of hypersensitivity reaction(s) that underlie the disorder. These reactions are typically classified into four types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immunity (CMI) reactions (also referred to as delayed-type hypersensitivity (DTH) reactions).
  • CMI cell-mediated immunity
  • DTH delayed-type hypersensitivity
  • Immunological diseases include inflammatory diseases and autoimmune diseases.
  • immunological diseases include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine opthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), anaphylaxis, allergic reaction, Sjogren's syndrome, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, xerophthalmia, conjunctivitis, Addison's disease, adrenalitis, thyroiditis
  • the use is for diabetic complications, type 1 diabetes, type 2 diabetes, diabetes disease progression, insulin resistance, atherosclerosis, intermittent claudication, peripheral vascular disease, aneurysm, acute coronary syndrome, heart failure, vascular inflammation, and myocardial infarction.
  • the immunological disorder is a T cell-mediated immunological disorder and accordingly, the anti-IL-1 B antibodies and agents as described herein are also useful for treating or preventing T cell-mediated immunological disorders.
  • the anti-IL-1 B antibodies or agents are useful for treating or preventing a respiratory disorder in which IL-1 B is abnormally expressed.
  • Treatment or prevention of the respiratory disorder is achieved by administering to a subject in need of such treatment or prevention an effective amount of the anti-IL-1 B antibody or agent, whereby the antibody inhibits the activity of IL-1 B associated with the disease state.
  • pulmonary emphysema of various origins
  • restrictive pulmonary diseases interstitial pulmonary diseases, interstitial lung disease, cystic fibrosis, bronchitis of various origins, bronchiectasis, ARDS (adult respiratory distress syndrome) and all forms of pulmonary oedema
  • obstructive pulmonary diseases selected from among COPD (chronic obstructive pulmonary disease), asthma, bronchial asthma, paediatric asthma, severe asthma, acute asthma attacks and chronic bronchitis
  • pulmonary emphysema which has its origins in COPD (chronic obstructive pulmonary disease) or a1 -proteinase inhibitor deficiency
  • restrictive pulmonary diseases selected from among allergic alveolitis, restrictive pulmonary diseases triggered by work-related noxious substances, such as asbestosis or silicosis, and restriction caused by lung tumours, such as lymphangio
  • the anti-IL-1 B antibodies and agents as described herein are also useful for treating cancers, in which IL-1 B is abnormally expressed.
  • IL-1 B-expressing cancers that can be treated by the methods described herein include, for example, leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (e.g., myeloblasts, promyelocytic, myelomonocytic, monocytic, or erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic) leukemia, or chronic lymphocytic leukemia; Polycythemia vera; Lymphoma (e.g., Hodgkin's disease or Non-Hodgkin's disease); myeloma, multiple myeloma, Waldenstrom's macroglobulinemia; heavy chain disease; solid tumors such sarcomas and carcinomas (e.g., fibrosarcoma
  • the anti-IL-1 B antibodies and agents as described herein are also useful for treating the following diseases: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, hyperuricaemia, familial Mediterranean fever, osteoarthritis, TNF receptor associated periodic syndrome, macular degeneration (in particular age-related, wet), acne, scleritis, neutrophilic dermatoses, muscle inflammation, autoimmune vestibular disorder,
  • compositions and Administration Thereof A composition comprising an IL-1 B binding agent (e.g., an anti-IL-1 B antibody) can be administered to a subject having or at risk of having an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation, or cancer.
  • an IL-1 B binding agent e.g., an anti-IL-1 B antibody
  • the invention further provides for the use of a IL-1 B binding agent (e.g., an anti-IL-1 B antibody) in the manufacture of a medicament for prevention or treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation, or cancer.
  • a IL-1 B binding agent e.g., an anti-IL-1 B antibody
  • CNS central nervous system
  • subject as used herein means any mammalian to which an IL-1 B binding agent can be administered, including, e.g., humans and non- human mammals, such as primates, rodents, and dogs. Subjects specifically intended for treatment using the methods described herein include humans.
  • the antibodies or agents can be administered either alone or in combination with other compositions in the prevention or treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation, or cancer.
  • Such compositions which can be administered in combination with the antibodies or agents include methotrexate (MTX) and immunomodulators, e.g. antibodies or small molecules.
  • MTX methotrexate
  • immunomodulators e.g. antibodies or small molecules.
  • antibodies for use in such pharmaceutical compositions are those that comprise an optimized antibody or antibody fragment having the light chain variable region amino acid sequence of any of SEQ ID NO: 25-33.
  • Examples of antibodies for use in such pharmaceutical compositions are also those that comprise an optimized antibody or antibody fragment having the heavy chain variable region amino acid sequence of any of SEQ ID NO: 34-40.
  • antibodies for use in such pharmaceutical compositions are also those that comprise an optimized antibody or antibody fragment having the light chain variable region and heavy chain variable region of any of SEQ ID NO: 25 and 34, SEQ ID NO: 27 and 35, SEQ ID NO: 27 and 36, SEQ ID NO: 26 and 37, SEQ ID NO: 27 and 38, SEQ ID NO: 28 and 40, SEQ ID NO: 29 and 39, SEQ ID NO: 30 and 40, SEQ ID NO: 31 and 40, SEQ ID NO: 32 and 40 or SEQ ID NO: 33 and 39.
  • IL-1 B binding agent can be administered, for example by infusion, bolus or injection, and can be administered together with other biologically active agents such as chemotherapeutic agents. Administration can be systemic or local. In preferred embodiments, the administration is by subcutaneous injection. Formulations for such injections may be prepared in for example prefilled syringes that may be administered once every other week.
  • the IL-1 B binding agent composition is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • a membrane such as a sialastic membrane, or a fiber.
  • the anti-IL-1 B antibody or agent is delivered in a controlled release system.
  • a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574).
  • polymeric materials can be used.
  • An IL-1 B binding agent e.g., an anti-IL-1 B antibody
  • An IL-1 B binding agent can be administered as pharmaceutical compositions comprising a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingredients.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to human beings.
  • compositions for administration by injection are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a IL-1 B binding agent (e.g., an anti-IL-1 B antibody) in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection.
  • a pharmaceutically acceptable diluent e.g., sterile water
  • the pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized anti-IL-1 B antibody or agent.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the amount of the IL-1 B binding agent (e.g., anti-IL-1 B antibody) that is effective in the treatment or prevention of an immunological disorder or cancer can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage of an anti-IL-1 B antibody or IL-1 B binding agent administered to a patient with an immunological disorder or IL-1 B-expressing cancer is typically about 0.1 mg/kg to about 100 mg/kg of the subject's body weight.
  • the dosage administered to a subject is about 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, or about 1 mg/kg to about 10 mg/kg of the subject's body weight.
  • Exemplary doses include, but are not limited to, from 1 ng/kg to 100 mg/kg.
  • a dose is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 1 1 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg or about 1 6 mg/kg.
  • the dose can be administered, for example, daily, once per week (weekly), twice per week, thrice per week, four times per week, five times per week, six times per week, biweekly or monthly, every two months, or every three months.
  • the dose is about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week, about 8 mg/kg/week, about 9 mg/kg/week, about 10 mg/kg/week, about 1 1 mg/kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week or about 1 6 mg/kg/week.
  • the dose ranges from about 1 mg/kg/week to about 15 mg/kg/week.
  • the pharmaceutical compositions comprising the IL-1 B binding agent can further comprise a therapeutic agent, either conjugated or unconjugated to the binding agent.
  • the anti-IL-1 B antibody or IL-1 B binding agent can be coadministered in combination with one or more therapeutic agents for the treatment or prevention of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, a disease of the central nervous system (CNS), for example a disease of the central nervous system (CNS) related to inflammation, or cancer.
  • Such combination therapy administration can have an additive or synergistic effect on disease parameters (e.g., severity of a symptom, the number of symptoms, or frequency of relapse).
  • an anti-IL-1 B antibody or IL-1 B binding agent is administered concurrently with a therapeutic agent.
  • the therapeutic agent is administered prior or subsequent to administration of the anti-IL-1 B antibody or IL-1 B binding agent, by at least an hour and up to several months, for example at least an hour, five hours, 12 hours, a day, a week, a month, or three months, prior or subsequent to administration of the anti-IL-1 B antibody or IL-1 B binding agent.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating the condition and may have a sterile access port.
  • the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • the active agent in the composition is the optimized anti-IL-1 B antibody.
  • the label on or associated with the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution, and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • mice were immunized with recombinant human IL-1 B and mouse hybridomas secreting anti-IL-1 B antibodies were generated according to standard procedures. Following this process, positive hits were analyzed in a cell based assay in which MH7A cells were stimulated with IL-1 B and the ability of antibodies to inhibit the phosphorylation of NFKB was monitored via Western blotting and the ability of these same antibodies to bind IL- 1 B was measured in a Luminex binding assay (Luminex Corporation, Austin, TX 78727, United States).
  • RNA was prepared from the hybridoma cells secreting these antibodies using a Qiagen (Hilden, Germany) RNeasy kit under standard procedures and this RNA was then reversed transcribed under standard procedures. This cDNA was then used as template DNA in a PCR amplification to amplify the sequences encoding the variable domains of these antibodies. These sequences were then synthesized and inserted into the expression vectors encoding human lgG1 constant domains enabling the assembly of these variable domains into human lgG1 molecules when both the heavy and light chains are co-expressed in mammalian cells. The recombinant antibodies were then harvested from the supernatant and purified to homogeneity using standard
  • the beads were then pelleted by centrifugation and blocked in PBS + 1 % BSA + 0.02% sodium azide for three hours at room temperature. Following this the beads were allowed to sediment and the supernatant was removed and the sediment was then resuspended in 1 ml PBS + 0.02% sodium azide and stored until use at 4°C.
  • IL-1 B concentration is diluted in assay buffer with constant 10 pM antibody as above to determine Kd as below:
  • a 10 pM AK antibody solution was prepared in Kinexa incubations buffer: 1 x PBS + 0,1 % BSA + 0,02% sodium azide and then hlL-1 B diluted in this buffer (1 :2 dilution series) from 1280 - 1 ,25 pM. These samples were then incubated at room temperature for 6 hours.
  • Kinetics and affinity of chimeric or optimized anti-IL1 B antibodies binding to recombinant human IL-1 are shown below (Table 1 1 ).
  • Kinetic binding data was determined using surface plasmon resonance (SPR) in the ProteOn XPR36 (Biorad, Hercules, CA).
  • a high surface density of Protein A/G (-6500RU) was immobilized over a GLM ProteOn sensor chip via direct amine coupling.
  • the anti-IL-1 B antibodies were captured over the protein A/G surface for the kinetic assay.
  • Recombinant human IL-1 B protein was prepared in PBS-T-EDTA buffer (Biorad, Hercules, CA) in the following concentration range:
  • Chimeric antibodies 36C2 and 47F1 1 were then profiled in a panel of cell based assays where the ability of these antibodies to inhibit the activity of cynomologus, mouse as well as human IL-1 B was determined (Table 12A).
  • 36C2 is a triple cross reactive antibody that is a potent inhibitor of human and cynomolgus IL-1 B activity and offers useful inhibitory activity against mouse IL-1 B activity.
  • 47F1 1 is a potent inhibitor of the activity of human and cynomolgus IL-1 B.
  • Optimized antibodies 36C2 and 47F1 1 were also profiled in a panel of cell based assays where the ability of these antibodies to inhibit the activity of cynomologus and human IL-1 B was determined (Table 12B).
  • Table 12A Table 12A
  • THP1 cells were stimulated with 100 nM PMA for 1 6 hours, washed with PBS and then incubated with fresh medium containing 100 g/ml E. coli lipopolysaccharide (01 1 1 :B4, Sigma Aldrich) for 7 hours.
  • PMA phorbol 12-myristate 13-acetate
  • MRC-5 cells 4000 cells per ml, EMEM (EBSS) (#M2279Sigma) + 10 % FCS (#A15-104 PAA)+ 1 x NEAA (#1 1 140 Invitrogen) + 2 mM Glutamine (#35050 Invitrogen)) were added to a well of a 96 well plate and incubated for 24 hours at 37°C, 5% C0 2 . After this time, the cells were stimulated with 1 1 pM human IL-1 B in the presence or absence of anti-IL-1 B antibodies. The cells were then incubated for a further 20 hours in the presence of IL-1 B and antibodies and after this time the supernatant was harvested.
  • the ability of the antibodies to inhibit the biological activity of IL-1 B was determined by measuring the concentration of IL-1 B stimulated IL-6 secretion in the supernatant which was determined using a commercially available IL-6 ELISA (BD OptEIA Set Human IL-6 ELISA, BD Biosciences, Heidelberg, Germany).
  • the original hybridoma antibody 36C2 was further profiled in a mouse endothelial bEnd3 cell line stimulated with 0.25 ng/ml (15 pM) recombinant mouse IL-1 B, resembling a condition where 80% of maximal keratinocyte chemoattractant (KC, a murine
  • 47F1 1 is a molecule that is capable of potently inhibiting the activity of human and cynomologus IL-1 B and can inhibit the activity of human IL-1 B at high concentrations of IL-1 B.
  • 36C2 is a molecule that is capable of potently inhibiting the activity of human and cynomologus IL-1 B and can inhibit the activity of mouse IL-1 B, which may be advantageous for in-vivo studies.
  • Recombinant chimeric and optimized anti-IL1 -B antibodies 36C2 and 47F1 1 were produced by transient transfection of CHO cells:
  • CHO-E cells growing in suspension in serum-free media were cultivated in shake flasks under agitation at 140rpm, 37C and 5% CO2 and kept at conditions of exponential growth rates.
  • cells were seeded at 1 -2x 10e6 cells/ml into 1 L of Gibco® FreeStyleTM CHO expression medium (LifeTechnologies, NY, US) and chemically transfected with 1 mg of light chain plasmid and 0.5mg of heavy chain plasmid. Cells were then incubated under orbital shaking for 10-12 days with one-time feeding of 150ml commercial feed solution to allow expression of the proteins.
  • Antibody titer in the cell culture supernatants were determined using an Octet® instrument (Pall ForteBio, CA, US) and protA biosensor tips according to manufacturer's instructions.
  • Mouse lead antibodies 36C2 and 47F1 1 were converted to chimeric antibodies consisting of the mouse variable domain of 36C2 and 47F1 1 and a human constant IgGI KO domain (see also Example 1 above). Sequences of mouse antibodies 36C2 and 47F1 1 are shown in Tables 1 and 2 herein above.
  • the IgG 1 KO knock out
  • the variable domains of the mouse and chimeric antibodies are identical. Chimeric antibodies are generated to confirm the function of the antibody and to ensure the correct sequence has been obtained.
  • the variable region of the antibody is then optimized through a design and screening process.
  • a library was made where human and mouse residues were varied in such a way that in any given position there could be either a human or mouse residue. Such a library was made for those amino acids that were different between human germline and mouse antibody.
  • the Epivax program was also used in the sequence optimization process.
  • the Epivax program is an in-siHco too ⁇ that predicts effector and regulatory T cell epitope content of proteins.
  • the presence of effector T cell epitopes in a protein is an important
  • the protein sequence is analysed in overlapping 9-mer peptide motifs and predictsif these peptides can bind to class II MHC haplotypes representing the overall human population, and finally by assessing if the peptide-MHC complex can stimulate activation of helper or regulatory T cells.
  • the algorithm has been refined over the years using experimentally generated data. Results are reported as Treg adjusted score, which subtracts those peptide-MHC complexes that stimulate regulatory T cells from those that stimulate effector/helper T cells. A lower Treg adjusted score predicts lower immunogenicity.

Abstract

La présente invention concerne des composés de liaison anti-IL-1B, en particulier des anticorps anti-IL-1B optimisés inédits, ainsi que des méthodes thérapeutiques et diagnostiques et des compositions permettant de les utiliser.
PCT/EP2015/065979 2014-07-14 2015-07-13 Anticorps anti-il-1b WO2016008851A1 (fr)

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US10919962B2 (en) 2012-02-13 2021-02-16 Agency For Science, Technology And Research Method of reducing tumor growth with IL-1beta neutralizing human monoclonal antibodies
JP2022506561A (ja) * 2018-11-07 2022-01-17 沢達生物医薬有限公司 ヒトＩＬ－１βに結合する抗体、その調製方法及び用途
WO2023044280A1 (fr) * 2021-09-20 2023-03-23 Eli Lilly And Company Anticorps anti-il-1-bêta
WO2023178265A3 (fr) * 2022-03-18 2023-10-26 Novascope Biochips Inc. Anticorps contre le virus du syndrome dysgénésique et respiratoire du porc et ses utilisations

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WO2004067568A2 (fr) * 2003-01-24 2004-08-12 Applied Molecular Evolution, Inc Antagonistes de l'il-1 beta humaine
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WO2002016436A2 (fr) * 2000-08-22 2002-02-28 Novartis Ag ANTICORPS DE LA IL-1β HUMAINE
US20030026806A1 (en) * 2000-10-27 2003-02-06 Amgen Inc. Antibodies and other selective IL-1 binding agents that allow binding to IL-1 receptor but not activation thereof
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Publication number Priority date Publication date Assignee Title
US10919962B2 (en) 2012-02-13 2021-02-16 Agency For Science, Technology And Research Method of reducing tumor growth with IL-1beta neutralizing human monoclonal antibodies
US11702471B2 (en) 2012-02-13 2023-07-18 Agency For Science, Technology And Research IL-1β neutralizing human monoclonal antibodies
US11780913B2 (en) 2012-02-13 2023-10-10 Agency For Science, Technology And Research IL-1β neutralizing human monoclonal antibodies
US11912761B2 (en) 2012-02-13 2024-02-27 Agency For Science, Technology And Research IL-1β neutralizing human monoclonal antibodies
JP2022506561A (ja) * 2018-11-07 2022-01-17 沢達生物医薬有限公司 ヒトＩＬ－１βに結合する抗体、その調製方法及び用途
EP3878867A4 (fr) * 2018-11-07 2022-07-06 Zeda Biopharmaceuticals, Inc. Anticorps se liant à l'il-1 ? humaine, son procédé de préparation et son utilisation
JP7256266B2 (ja) 2018-11-07 2023-04-11 沢達生物医薬有限公司 ヒトＩＬ－１βに結合する抗体、その調製方法及び用途
WO2023044280A1 (fr) * 2021-09-20 2023-03-23 Eli Lilly And Company Anticorps anti-il-1-bêta
WO2023178265A3 (fr) * 2022-03-18 2023-10-26 Novascope Biochips Inc. Anticorps contre le virus du syndrome dysgénésique et respiratoire du porc et ses utilisations

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