WO2021028752A1 - Anticorps anti-tfn pour le traitement du diabète de type i - Google Patents

Anticorps anti-tfn pour le traitement du diabète de type i Download PDF

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Publication number
WO2021028752A1
WO2021028752A1 PCT/IB2020/057066 IB2020057066W WO2021028752A1 WO 2021028752 A1 WO2021028752 A1 WO 2021028752A1 IB 2020057066 W IB2020057066 W IB 2020057066W WO 2021028752 A1 WO2021028752 A1 WO 2021028752A1
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Prior art keywords
tnf
patient
antibody
administering
peptide
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PCT/IB2020/057066
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English (en)
Inventor
Joseph Hedrick
Elizabeth C. Hsia
Paul IMM
Jocelyn LEU
Bethany PAXSON
Mark Rigby
Songmao ZHENG
Ramineh ZOKA
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Janssen Biotech, Inc.
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Publication of WO2021028752A1 publication Critical patent/WO2021028752A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a fde name “JBI6138WOPCTlSequenceListing.txt”, creation date of July 22, 2020 and having a size of 26kb.
  • the sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
  • the present invention relates to materials and methods for treating Type 1 Diabetes (T1D) with a TNF inhibitor, e.g., wherein the TNF inhibitor is the anti-TNF antibody SIMPONI® (golimumab) having a heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37 or an antigen binding fragment thereof.
  • TNF inhibitor is the anti-TNF antibody SIMPONI® (golimumab) having a heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37 or an antigen binding fragment thereof.
  • TNF alpha is a soluble homotrimer of 17 kD protein subunits.
  • a membrane- bound 26 kD precursor form of TNF also exists.
  • TNF alpha Cells other than monocytes or macrophages also produce TNF alpha.
  • human non-monocytic tumor cell lines produce TNF alpha and CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines also produce TNF alpha.
  • TNF alpha causes pro-inflammatory actions which result in tissue injury, such as degradation of cartilage and bone, induction of adhesion molecules, inducing procoagulant activity on vascular endothelial cells, increasing the adherence of neutrophils and lymphocytes, and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells.
  • TNF alpha has been associated with infections, immune disorders, neoplastic pathologies, autoimmune pathologies and graft-versus-host pathologies.
  • the association of TNF alpha with cancer and infectious pathologies is often related to the host's catabolic state. Cancer patients suffer from weight loss, usually associated with anorexia.
  • Cachexia The extensive wasting which is associated with cancer, and other diseases, is known as "cachexia". Cachexia includes progressive weight loss, anorexia, and persistent erosion of lean body mass in response to a malignant growth. The cachectic state causes much cancer morbidity and mortality. There is evidence that TNF alpha is involved in cachexia in cancer, infectious pathology, and other catabolic states.
  • TNF alpha is believed to play a central role in gram-negative sepsis and endotoxic shock, including fever, malaise, anorexia, and cachexia.
  • Endotoxin strongly activates monocyte/macrophage production and secretion of TNF alpha and other cytokines.
  • TNF alpha and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin.
  • Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release. Circulating TNF alpha increases in patients suffering from Gram-negative sepsis.
  • TNF alpha has been implicated in inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignancies, and/or neurodegenerative diseases and is a useful target for specific biological therapy in diseases, such as rheumatoid arthritis and Crohn's disease.
  • beneficialal effects in open- label trials with a chimeric monoclonal antibody to TNF alpha (cA2) have been reported with suppression of inflammation and with successful retreatment after relapse in rheumatoid arthritis and in Crohn's disease.
  • Beneficial results in a randomized, double-blind, placebo-controlled trial with cA2 have also been reported in rheumatoid arthritis with suppression of inflammation.
  • mAbs specific for recombinant human TNF which had neutralizing activity in vitro. Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays and to assist in the purification of recombinant TNF. However, these studies do not provide a basis for producing TNF neutralizing antibodies that can be used for in vivo diagnostic or therapeutic uses in humans, due to immunogenicity, low specificity and/or pharmaceutical unsuitability.
  • Neutralizing antisera or mAbs to TNF have been shown in mammals other than man to abrogate adverse phaysiological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, e.g., in rodent lethality assays and in primate pathology model systems.
  • Putative receptor binding loci of hTNF has been disclosed and the receptor binding loci of TNF alpha as consisting of amino acids 11-13, 37-42, 49-57 and 155- 157 of TNF have been disclosed.
  • Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/or monoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestion or fusion protein products thereof) are potential therapeutic agents that are being investigated in some cases to attempt to treat certain diseases.
  • Such antibodies or fragments can elicit an immune response when administered to humans.
  • Such an immune response can result in an immune complex-mediated clearance of the antibodies or fragments from the circulation, and make repeated administration unsuitable for therapy, thereby reducing the therapeutic benefit to the patient and limiting the readministration of the antibody or fragment.
  • repeated administration of antibodies or fragments comprising non-human portions can lead to serum sickness and/or anaphylaxis.
  • TNF inhibitors that overcame one more of these problems led to development of currently marketed anti-TNF antibodies and other TNF inhibitors, e.g., anti-TNF antibodies such as REMICADE® (infliximab), HUMIRA® (adalimumab), and SIMPONI® (golimumab).
  • Other TNF inhibitors include, e.g., CIMZIA® (certolizumab pegol), a PEGylated antibody fragment, and ENBREL® (etanercept), a soluble TNF receptor fusion protein.
  • CIMZIA® certolizumab pegol
  • ENBREL® etanercept
  • TNF inhibitors see, e.g., Lis et al., Arch MedSci. 2014 Dec 22; 10(6): 1175-1185.
  • Type 1 diabetes mellitus also known as autoimmune diabetes
  • T1DM or T1D Type 1 diabetes mellitus
  • T1D also known as autoimmune diabetes
  • T1D is a chronic disease characterized by insulin deficiency due to pancreatic b-cell loss that leads to hyperglycemia.
  • T1D the pathogenesis of the disease is thought to involve T cell-mediated destruction of b-cells.
  • Islet-targeting autoantibodies that target insulin, 65 kDa glutamic acid decarboxylase, insulinoma-associated protein 2 and zinc transporter 8, all of which are proteins associated with secretory- granules in b-cells, are biomarkers of TIDM-associated autoimmunity that are found months to years before symptom onset, and can be used to identify and study individuals who are at risk of developing T1D.
  • the pathogenesis of T1D can be divided into three stages depending on the absence or presence of hyperglycemia and hyperglycemia-associated symptoms (such as polyuria and thirst).
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor.
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor, wherein the patient in need of treatment to preserve beta-cell function is selected
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor, wherein the minimal increase in the fasting proinsulin-to-C
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody or an anti
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor.
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the determining of the fasting proinsulin-to- C
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a patient diagnosed with pre-Type
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a newly diagnosed T1D patient
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a patient diagnosed with T1D
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody or an anti
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising
  • the present invention provides a method of treatment to control proinsulin-to-C-peptide ratio in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor.
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the determining of the fasting proinsulin-to- C-peptide ratio after administering the TNF inhibitor is at 12
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a patient diagnosed with pre-Type 1 Diabetes (pre-TID), wherein pre-TID
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a newly diagnosed T1D patient, wherein the newly diagnosed T1D patient is diagnosed within about
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the patient in need thereof is a patient diagnosed with T1D, and wherein the patient has a stimulated peak C-
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody or an antigen binding fragment thereof.
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising a heavy chain (HC) having the amino acid sequence of SEQ
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising a heavy chain (HC) having the amino acid sequence of SEQ
  • the present invention provides a method of treatment to preserve beta-cell function in a human patient in need thereof, the method comprising: a.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining that the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or the increase in the fasting proinsulin-to-C-peptide ratio is ⁇ 5% after administering the TNF inhibitor, wherein the TNF inhibitor is an anti-TNF antibody comprising a heavy chain (HC) having the amino acid sequence of SEQ
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient an isolated anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P- value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C-peptide concentration following a mixed-
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient an isolated anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P- value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C-peptide concentration following a mixed-
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient an isolated anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P- value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C-peptide concentration following a mixed-
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient a composition comprising an isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P-value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient a composition comprising an isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P-value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising, administering to the patient a composition comprising an isolated mammalian anti-TNF antibody having a heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:36 and a light chain (LC) comprising an amino acid sequence of SEQ ID NO:37, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the anti-TNF antibody has a statistically significant (P-value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C
  • the present invention provides a method to restore beta cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to restore beta-cell function; b.) determining a stimulated peak C- peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptid
  • the present invention provides a method to restore beta cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to restore beta-cell function; b.) determining a stimulated peak C- peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptid
  • the present invention provides a method to restore beta cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to restore beta-cell function; b.) determining a stimulated peak C- peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptid
  • the present invention provides a method to restore beta cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to restore beta-cell function; b.) determining a stimulated peak C- peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; c.) administering the TNF inhibitor to the patient; d.) determining the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; e.) comparing the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; f.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptid
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method of treatment to restore beta-cell function in a human patient in need thereof, the method comprising: a.) determining a stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a TNF inhibitor to the patient; b.) administering the TNF inhibitor to the patient; c.) determining the stimulated peak C-peptide concentration following a mixed-meal tolerance test (MMTT) and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the TNF inhibitor; d.) comparing the stimulated peak C- peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor to the patient; e.) determining the beta-cell function is restored in the patient if there is an increase in stimulated peak C-peptide concentration following a MMTT and/or
  • the present invention provides a method to preserve beta-cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to preserve beta-cell function; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a means for inhibiting TNF to the patient; c.) administering the means for inhibiting TNF to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the means for inhibiting TNF; e.) comparing the fasting proinsulin-to-C-peptide ratio determined before and after administering the means for inhibiting TNF to the patient; f.) determining the beta-cell function is preserved in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the means for inhibiting TNF or there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the means for inhibiting T
  • the present invention provides a method to control proinsulin-to-C-peptide ratio in a human patient, the method comprising: a.) selecting a human patient in need of treatment to control proinsulin-to-C-peptide ratio; b.) determining a fasting proinsulin-to-C-peptide ratio of the patient before administering a means for inhibiting TNF to the patient; c.) administering the means for inhibiting TNF to the patient; d.) determining the fasting proinsulin-to-C-peptide ratio of the patient after administering the means for inhibiting TNF; e.) comparing the fasting proinsulin- to-C-peptide ratio determined before and after administering the means for inhibiting TNF to the patient; f.) determining the proinsulin-to-C-peptide ratio is controlled in the patient if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the means for inhibiting TNF or there is a minimal increase in the fast
  • the present invention provides a method for treating a TNF related condition in a human patient, wherein the TNF related condition is newly diagnosed Type 1 Diabetes (T1D), wherein the newly diagnosed T1D is diagnosed within about 100 days before receiving treatment, the method comprising: administering to the patient a means for inhibiting TNF, wherein the patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test (MMTT), and wherein the patient treated with the means for inhibiting TNF has a statistically significant (P-value ⁇ 0.05) difference compared to placebo in one or more criteria selected from the group consisting of: change from baseline in insulin use, hypoglycemia event rate, stimulated peak C-peptide concentration following a mixed- meal tolerance test (MMTT), and fasting proinsulin-to-C-peptide ratio.
  • T1D Type 1 Diabetes
  • MMTT mixed-meal tolerance test
  • the present invention provides a method to restore beta cell function in a human patient, the method comprising: a.) selecting a human patient in need of treatment to restore beta-cell function; b.) determining a stimulated peak C- peptide concentration following a mixed-meal tolerance test (MMTT) and/or a fasting proinsulin-to-C-peptide ratio of the patient before administering a means for inhibiting TNF to the patient; c.) administering the means for inhibiting TNF to the patient; d.) determining the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio of the patient after administering the means for inhibiting TNF; e.) comparing the stimulated peak C-peptide concentration following a MMTT and/or the fasting proinsulin-to-C-peptide ratio determined before and after administering the means for inhibiting TNF to the patient; f.) determining the beta-cell function is restored in the patient
  • a patient diagnosed with pre-TID refers to a patient prior to the onset of symptoms in the patient and requirement for exogenous insulin, and wherein the patient is 6 to 21 years of age and has >2 auto-antibodies for T1D.
  • a “newly diagnosed T1D patient” refers to a patient diagnosed within about 100 days before receiving treatment, wherein the newly diagnosed T1D patient is 6 to 21 years of age, has >1 auto-antibodies for T1D, and has a stimulated peak C-peptide concentration of >0.2 pmol/mL following a mixed-meal tolerance test.
  • diagnosisd within about 100 days before receiving treatment refers to being diagnosed within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
  • not an increase” in the fasting proinsulin-to-C-peptide ratio includes a decrease and no significant change in the fasting proinsulin-to-C-peptide ratio.
  • a “minimal increase” in the fasting proinsulin-to-C-peptide ratio is an increase of ⁇ 1%, ⁇ 2%, ⁇ 3%, ⁇ 4%, or ⁇ 5%.
  • TNF inhibitor is a is a pharmaceutical product that inhibits the physiologic response to tumor necrosis factor (TNF), i.e., inhibits TNF activity.
  • TNF inhibitors includes e.g., antibodies such as REMICADE® (infliximab), HUMIRA® (adalimumab), and SIMPONI® (golimumab).
  • Other TNF inhibitors include, e.g., CIMZIA® (certolizumab pegol), a PEGylated antibody fragment, and ENBREL® (etanercept), a soluble TNF receptor fusion protein.
  • determining fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor is after there has been time for the TNF inhibitor to effect TNF activity and produce a measurable change in beta-cell function as determined by the fasting proinsulin-to-C-peptide ratio, e.g., “after administering the TNF inhibitor” is after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • FIG. 1 shows a graphical representation showing an assay for ability of TNV mAbs in hybridoma cell supernatants to inhibit TNFa binding to recombinant TNF receptor.
  • Varying amounts of hybridoma cell supernatants containing known amounts of TNV mAh were preincubated with a fixed concentration (5 ng/ml) of 125 I-labeled TNFa. The mixture was transferred to 96-well Optiplates that had been previously coated with p55-sf2, a recombinant TNF receptor/IgG fusion protein. The amount of TNFa that bound to the p55 receptor in the presence of the mAbs was determined after washing away the unbound material and counting using a gamma counter.
  • TNV mAb samples were tested in these experiments, for simplicity three of the mAbs that were shown by DNA sequence analyses to be identical to one of the other TNV mAbs (see Section 5.2.2) are not shown here. Each sample was tested in duplicate. The results shown are representative of two independent experiments.
  • FIGS. 2A-B shows DNA sequences of the TNV mAb heavy chain variable regions.
  • the germline gene shown is the DP-46 gene.
  • 'TNVs' indicates that the sequence shown is the sequence of TNV14, TNV15, TNV148, and TNV196.
  • the first three nucleotides in the TNV sequence define the translation initiation Met codon.
  • Dots in the TNV mAb gene sequences indicate the nucleotide is the same as in the germline sequence.
  • the first 19 nucleotides (underlined) of the TNV sequences correspond to the oligonucleotide used to PCR-amplify the variable region.
  • An amino acid translation (single letter abbreviations) starting with the mature mAb is shown only for the germline gene.
  • TNV148(B) The three CDR domains in the germline amino acid translation are marked in bold and underlined. Lines labeled TNV148(B) indicate that the sequence shown pertains to both TNV148 and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to the sequence not being known or not existing in the germline gene.
  • the TNV mAb heavy chains use the J6 joining region.
  • FIG. 3 shows DNA sequences of the TNV mAb light chain variable regions.
  • the germline gene shown is a representative member of the Vg/38K family of human kappa germline variable region genes. Dots in the TNV mAb gene sequences indicate the nucleotide is the same as in the germline sequence.
  • the first 16 nucleotides (underlined) of the TNV sequences correspond to the oligonucleotide used to PCR-amplify the variable region.
  • An amino acid translation of the mature mAb (single letter abbreviations) is shown only for the germline gene. The three CDR domains in the germline amino acid translation are marked in bold and underlined.
  • FIG. 4 shows deduced amino acid sequences of the TNV mAh heavy chain variable regions. The amino acid sequences shown (single letter abbreviations) were deduced from DNA sequence determined from both uncloned PCR products and cloned PCR products. The amino sequences are shown partitioned into the secretory signal sequence (signal), framework (FW), and complementarity determining region (CDR) domains.
  • signal secretory signal sequence
  • FW framework
  • CDR complementarity determining region
  • the amino acid sequence for the DP-46 germline gene is shown on the top line for each domain. Dots indicate that the amino acid in the TNV mAh is identical to the germline gene. TNV 148(B) indicates that the sequence shown pertains to both TNV148 and TNV148B. 'TNVs' indicates that the sequence shown pertains to all TNV mAbs unless a different sequence is shown. Dashes in the germline sequence (CDR3) indicate that the sequences are not known or do not exist in the germline gene.
  • FIG. 5 shows deduced amino acid sequences of the TNV mAh light chain variable regions.
  • the amino acid sequences shown (single letter abbreviations) were deduced from DNA sequence determined from both uncloned PCR products and cloned PCR products.
  • the amino sequences are shown partitioned into the secretory signal sequence (signal), framework (FW), and complementarity determining region (CDR) domains.
  • the amino acid sequence for the Vg/38K-type light chain germline gene is shown on the top line for each domain. Dots indicate that the amino acid in the TNV mAh is identical to the germline gene.
  • TNV148(B) indicates that the sequence shown pertains to both TNV148 and TNV148B. All' indicates that the sequence shown pertains to TNV 14, TNV15, TNV148, TNV148B, and TNV186.
  • FIG. 6 shows schematic illustrations of the heavy and light chain expression plasmids used to make the rTNV148B-expressing C466 cells.
  • pl783 is the heavy chain plasmid and pl776 is the light chain plasmid.
  • the rTNV148B variable and constant region coding domains are shown as black boxes.
  • the immunoglobulin enhancers in the J-C introns are shown as gray boxes. Relevant restriction sites are shown.
  • the plasmids are shown oriented such that transcription of the Ab genes proceeds in a clockwise direction.
  • Plasmid pl783 is 19.53 kb in length and plasmid pl776 is 15.06 kb in length. The complete nucleotide sequences of both plasmids are known.
  • variable region coding sequence in pl783 can be easily replaced with another heavy chain variable region sequence by replacing the BsiWI/BstBI restriction fragment.
  • variable region coding sequence in pi 776 can be replaced with another variable region sequence by replacing the Sall/Aflll restriction fragment.
  • FIG. 7 shows graphical representation of growth curve analyses of five rTNV148B-producing cell lines. Cultures were initiated on day 0 by seeding cells into T75 flasks in I5Q+MHX media to have a viable cell density of 1.0 X 10 5 cells/ml in a 30 ml volume. The cell cultures used for these studies had been in continuous culture since transfections and subclonings were performed. On subsequent days, cells in the T flasks were thoroughly resuspended and a 0.3 ml aliquot of the culture was removed. The growth curve studies were terminated when cell counts dropped below 1.5 X 10 5 cells/ml. The number of live cells in the aliquot was determined by trypan blue exclusion and the remainder of the aliquot stored for later mAb concentration determination. An ELISA for human IgG was performed on all sample aliquots at the same time.
  • FIG. 8 shows a graphical representation of the comparison of cell growth rates in the presence of varying concentrations of MHX selection.
  • Cell subclones C466A and C466B were thawed into MHX-free media (IMDM, 5% FBS, 2 mM glutamine) and cultured for two additional days. Both cell cultures were then divided into three cultures that contained either no MHX, 0.2X MHX, or IX MHX.
  • IMDM 5% FBS, 2 mM glutamine
  • FIG. 9 shows graphical representations of the stability of mAb production over time from two rTNV148B-producing cell lines.
  • Cell subclones that had been in continuous culture since performing transfections and subclonings were used to start long-term serial cultures in 24-well culture dishes.
  • Cells were cultured in I5Q media with and without MHX selection.
  • Cells were continually passaged by splitting the cultures every 4 to 6 days to maintain new viable cultures while previous cultures were allowed to go spent. Aliquots of spent cell supernatant were collected shortly after cultures were spent and stored until the mAb concentrations were determined.
  • An ELISA for human IgG was performed on all sample aliquots at the same time.
  • mice 10 shows arthritis mouse model mice Tg 197 weight changes in response to anti-TNF antibodies of the present invention as compared to controls in Example 4.
  • Tgl97 study mice were assigned, based on gender and body weight, to one of 9 treatment groups and treated with a single intraperitoneal bolus dose of Dulbecco’s PBS (D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148 or TNV196) at either 1 mg/kg or 10 mg/kg.
  • D-PBS Dulbecco’s PBS
  • TNV14, TNV148 or TNV196 anti-TNF antibody of the present invention
  • the animals treated with 10 mg/kg cA2 showed consistently higher weight gain than the D-PBS-treated animals throughout the study. This weight gain was significant at weeks 3-7.
  • the animals treated with 10 mg/kg TNV148 also achieved significant weight gain at week 7 of the study.
  • FIGS. 11A-C represent the progression of disease severity based on the arthritic index as presented in Example 4.
  • the 10 mg/kg cA2 -treated group’s arthritic index was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7).
  • the animals treated with 1 mg/kg TNV14 and the animals treated with 1 mg/kg cA2 failed to show significant reduction in AI after week 3 when compared to the D-PBS-treated Group.
  • the 1 mg/kg TNV148 showed a significantly lower AI than 1 mg/kg cA2 at 3, 4 and 7 weeks.
  • the 1 mg/kg TNV148 was also significantly lower than the 1 mg/kg TNV14-treated Group at 3 and 4 weeks.
  • TNV196 showed significant reduction in AI up to week 6 of the study (when compared to the D-PBS- treated Group), TNV148 was the only 1 mg/kg treatment that remained significant at the conclusion of the study.
  • FIG. 12 shows arthritis mouse model mice Tg 197 weight changes in response to anti-TNF antibodies of the present invention as compared to controls in Example 5.
  • Tgl97 study mice were assigned, based on body weight, to one of 8 treatment groups and treated with a intraperitoneal bolus dose of control article (D-PBS) or antibody (TNV14, TNV148) at 3 mg/kg (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for test article efficacy. Serum samples, obtained from animals in Groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4.
  • D-PBS control article
  • TNV14, TNV148 antibody
  • FIGS. 13A-C are graphs representing the progression of disease severity in Example 5 based on the arthritic index.
  • the 10 mg/kg cA2 -treated group’s arthritic index was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5).
  • the animals treated with 1 mg/kg or 3 mg/kg of cA2 and the animals treated with 3 mg/kg TNV14 failed to achieve any significant reduction in AI at any time throughout the study when compared to the d-PBS control group.
  • the animals treated with 3 mg/kg TNV148 showed a significant reduction when compared to the d-PBS-treated group starting at week 3 and continuing through week 5.
  • the 10 mg/kg cA2 -treated animals showed a significant reduction in AI when compared to both the lower doses ( 1 mg/kg and 3 mg/kg) of cA2 at weeks 4 and 5 of the study and was also significantly lower than the TNV14-treated animals at weeks 3-5. Although there appeared to be no significant differences between any of the 3mg/kg treatment groups, the AI for the animals treated with 3 mg/kg TNV14 were significantly higher at some time points than the 10 mg/kg whereas the animals treated with TNV148 were not significantly different from the animals treated with 10 mg/kg of cA2.
  • FIG. 14 shows arthritis mouse model mice Tg 197 weight changes in response to anti-TNF antibodies of the present invention as compared to controls in Example 6.
  • Tgl97 study mice were assigned, based on gender and body weight, to one of 6 treatment groups and treated with a single intraperitoneal bolus dose of antibody (cA2, or TNV148) at either 3 mg/kg or 5 mg/kg.
  • This study utilized the D-PBS and 10 mg/kg cA2 control Groups.
  • FIG. 15 represents the progression of disease severity based on the arthritic index as presented in Example 6. All treatment groups showed some protection at the earlier time points, with the 5 mg/kg cA2 and the 5 mg/kg TNV148 showing significant reductions in AI at weeks 1-3 and all treatment groups showing a significant reduction at week 2. Later in the study the animals treated with 5 mg/kg cA2 showed some protection, with significant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) of both the cA2 and the TNV148 showed significant reductions at 6 and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain a significant reduction at the conclusion of the study (week 8). There were no significant differences between any of the treatment groups (excluding the saline control group) at any time point.
  • FIG. 16 shows arthritis mouse model mice Tg 197 weight changes in response to anti-TNF antibodies of the present invention as compared to controls in Example 7.
  • TNV148 derived from hybridoma cells
  • rTNV148B derived from transfected cells
  • FIG. 17 represents the progression of disease severity based on the arthritic index as presented in Example 7.
  • the 10 mg/kg cA2 -treated group’s arthritic index was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8).
  • Both of the TNV148-treated Groups and the 1 mg/kg cA2 -treated Group showed a significant reduction in AI at week 4.
  • a previous study (P-099-017) showed that TNV148 was slightly more effective at reducing the Arthritic Index following a single 1 mg/kg intraperitoneal bolus, this study showed that the AI from both versions of the TNV antibody-treated groups was slightly higher.
  • FIG. 18 shows study schema for study CNT0148DML2001.
  • FIG. 19 shows simulated golimumab concentrations (median and 95% Prediction Interval) through Week 24 for various dosing regimens (without MTX) in virtual patients ages 6 - 21.
  • Panel A T1D (red) vs JIA (blue) and
  • Panel B T1D (red) vs pedUC (green).
  • FIG. 20 shows simulated Time Course for Free TNFa Suppression with Induction Period (1st month) in the left panel and Maintenance Period (through 6 months) in the right panel.
  • FIG. 21 shows features of ULTRASAFE PASSIVE® (needle guard).
  • FIG. 22 shows an illustration of SIMPONI® (golimumab) VARIOJECT® (injector) features.
  • FIG. 23 shows steps in the administration of SIMPONI® (golimumab) using the VARIOJECT® (injector) device.
  • FIG. 24 shows proposed design modifications to VARIOJECT® (injector) device.
  • FIG. 25 shows least squares Mean (LSMean) change from baseline in C- peptide AUC in log(AUC+l) scale for MMTT-stimulated 4-hour C-peptide AUC (pmol/mL) in patients treated with golimumab or placebo. *p ⁇ 0.05
  • FIG. 26 shows treatment difference in 4-hour C-peptide AUC at week 52 in log(AUC+l) scale for primary and sensitivity analyses. All analyses are baseline characteristics adjusted. AUC, area under the curve; MMRM, mixed model for repeated measures; ANCOVA, analysis of covariance; LS, least squares; Cl, confidence interval; cLA, constrained longitudinal analysis.
  • Primary is the primary analysis using a mixed model for repeated measures (MMRM).
  • Per-protocol (MMRM) is the same model as in the primary analysis on per-protocol population that is defined as the full analysis set with exclusion of participants with major protocol deviations that may potentially confound the primary endpoint.
  • effect linear is to fit a random effect model on log(AUC+l) at all time points (including baseline) as the response variable, age, gender, time (continuous variable), treatment and treatment by time interaction as fixed effects, interception and slope as random effects imputation is to apply an ANCOVA model with multiple imputation as missing data handling strategy.
  • ⁇ ANCOVA linear extrapolation is to perform an ANCOVA model with log(AUC+l) at Week 52 as the response variable, baseline log(AUC+1), gender and age as covariates, and treatment as a fixed factor. Missing data handling strategy of linear extrapolation is implemented.
  • cLA MMRM refers to constrained longitudinal analysis that applies log(AUC+l) including baseline as the response variable, the fixed, categorical effects of treatment, time, and treatment-by-time interaction, as well as the fixed covariate of age.
  • FIG. 27 shows LSMean and Standard Error (SE) for change from baseline in HbAlc (%) Over Time.
  • FIG. 28 shows results for post-hoc analysis of treatment difference in change from baseline in HbAlc at week 52. All analyses are baseline characteristics (age, gender, and baseline) adjusted.
  • FIGS. 29A-B show proportions and treatment differences of C Responders, R responders, and C+R Responders.
  • FIG.29A shows proportions of responders by treatment (Golimumab and Placebo).
  • FIG. 29B shows treatment differences (Golimumab and Placebo) for different responders. The 95% confidence interval was based on Wilson score method.
  • FIGS. 30A-B show proportions and treatment differences of subjects achieving HbAlc goals at week 52.
  • FIG.30A shows proportions subjects achieving HbAlc goals at week 52 by treatment (Golimumab and Placebo).
  • FIG. 3 OB shows treatment differences (Golimumab and Placebo) for subjects achieving HbAlc goals at week 52.
  • the 95% confidence interval was based on Wilson score method.
  • FIG. 31 shows change in median fasting proinsulin-to-C-peptide ratio (%) over time for treatment with golimumab and placebo. *Nominal p ⁇ 0.0001.
  • FIG. 32 shows change in daily insulin use (U/day) over time for treatment with golimumab and placebo.
  • FIG. 33 shows proportion (%) of subjects with an increase or minimal decrease in 4-hour C-peptide AUC for treatment with golimumab and placebo, minimal decrease in C-peptide AUC is defined as ⁇ 5% from baseline.
  • FIG. 34 shows proportion (%) of subjects in partial remission of T1D overtime for treatment with golimumab and placebo. Partial remission is defined as an insulin dose-adjusted Ale (IDAAlc) of ⁇ 9.
  • FIG. 35 shows least squares Mean (LSMean) change from baseline in C- peptide AUC in log(AUC+l) scale for MMTT-stimulated 2-hour C-peptide AUC (pmol/mL) in patients treated with golimumab or placebo.
  • LSMean least squares Mean
  • FIG. 36A-B show scatterplots comparing 2-hour versus 4-hour MMTT at (FIG. 36 A) Baseline and (FIG. 36B) Week 52.
  • FIG. 37A-B show bar graphs corresponding to percent of subjects achieving C Responder (FIG. 37A), C+R Responder (FIG. 37B), R Responder (FIG. 37C), and Non Responder (FIG. 37D) status at Week 52 (+90% Cl) grouped by different serum golimumab concentration quartiles (mg/mL) at Week 52.
  • the present invention relates to compositions and methods of treatment to preserve b-cell function with a TNF inhibitor.
  • TNF inhibitor tumor necrosis factor
  • anti-TNF antibodies such as REMICADE® (infliximab), HUMIRA® (adalimumab), and SIMPONI® (golimumab).
  • TNF inhibitors include, e.g., CIMZIA® (certolizumab pegol), a PEGylated antibody fragment, and ENBREL® (etanercept), a soluble TNF receptor fusion protein.
  • CIMZIA® certolizumab pegol
  • PEGylated antibody fragment etanercept
  • ENBREL® etanercept
  • the present invention provides isolated, recombinant and/or synthetic anti-TNF human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted, antibodies comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2 and 3 and/or all of the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 and TNF anti-idiotype antibodies thereto, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one anti-TNF antibody or anti-idiotype antibody.
  • the present invention further includes, but is not limited to, methods of making and using such nucleic acids and antibodies and anti idiotype antibodies, including diagnostic and therapeutic compositions, methods and devices.
  • an "anti-tumor necrosis factor alpha antibody,” “anti-TNF antibody,” “anti-TNF antibody portion,” or “anti-TNF antibody fragment” and/or “anti- TNF antibody variant” and the like include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an TNF receptor or binding protein, which can be incorporated into an antibody of the present invention.
  • CDR complementarity determining region
  • Such antibody optionally further affects a specific ligand, such as but not limited to where such antibody modulates, decreases, increases, antagonizes, agonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with at least one TNF activity or binding, or with TNF receptor activity or binding, in vitro, in situ and/or in vivo.
  • a suitable anti-TNF antibody, specified portion or variant of the present invention can bind at least one TNF, or specified portions, variants or domains thereof.
  • a suitable anti-TNF antibody, specified portion, or variant can also optionally affect at least one of TNF activity or function, such as but not limited to, R A, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
  • TNF activity or function such as but not limited to, R A, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
  • Functional fragments include antigen-binding fragments that bind to a mammalian TNF.
  • antibody fragments capable of binding to TNF or portions thereof including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab’)2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc’ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).
  • Fab e.g., by papain digestion
  • Fab' e.g., by pepsin digestion and partial reduction
  • F(ab’)2 e.g., by pepsin digestion
  • facb e.g., by plasmin digestion
  • Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and/or hinge region of the heavy chain.
  • the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CHI, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • antibodies designated primate monkey, baboon, chimpanzee, etc.
  • rodent mouse, rat, rabbit, guinea pig, hamster, and the like
  • other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
  • chimeric antibodies include any combination of the above.
  • a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for at least one TNF protein, the other one is for any other antigen.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)).
  • Anti-TNF antibodies useful in the methods and compositions of the present invention can optionally be characterized by high affinity binding to TNF and optionally and preferably having low toxicity.
  • an antibody, specified fragment or variant of the invention, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity is useful in the present invention.
  • the antibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Fow or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results achieved.
  • “Fow immunogenicity” is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titers in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al., Lancet 344: 1125-1127 (1994), entirely incorporated herein by reference).
  • the isolated nucleic acids of the present invention can be used for production of at least one anti-TNF antibody or specified variant thereof, which can be used to measure or effect in an cell, tissue, organ or animal (including mammals and humans), to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least one TNF condition, selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic disorder or disease.
  • Such a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms.
  • the effective amount can comprise an amount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple or continuous administration, or to achieve a serum concentration of 0.01-5000 mg/ml serum concentration per single, multiple, or continuous administration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts.
  • Citations. All publications or patents cited herein are entirely incorporated herein by reference as they show the state of the art at the time of the present invention and/or to provide description and enablement of the present invention. Publications refer to any scientific or patent publications, or any other information available in any media format, including all recorded, electronic or printed formats.
  • At least one anti-TNF antibody of the present invention comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2 and 3 and/or all of the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art.
  • Human antibodies that are specific for human TNL proteins or fragments thereof can be raised against an appropriate immunogenic antigen, such as isolated and/or TNL protein or a portion thereof (including synthetic molecules, such as synthetic peptides). Other specific or general mammalian antibodies can be similarly raised. Preparation of immunogenic antigens, and monoclonal antibody production can be performed using any suitable technique.
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SSI, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art.
  • a suitable immortal cell line e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS
  • antibody producing cells such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra
  • Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention.
  • the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK; Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, CA; Ixsys.
  • a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK
  • single cell antibody producing technologies e.g., selected lymphocyte antibody method (“SLAM”) (US pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell et al., Biotechnok 8:333-337 (1990); One Cell Systems, Cambridge, MA; Gray et al., J. Imm. Meth.
  • SLAM selected lymphocyte antibody method
  • a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence.
  • baserv.uci.kun.nl/ ⁇ jraats/linksl .html www. recab.uni-hd.de/immuno.bme.nwu.edu/; www . mrc-cpe .cam .ac .uk/imt-doc/public/INTRO .html ; www. ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr: 8104/; www. biochem.ucl.ac.uk/ ⁇ martin/abs/ index.html; antibody.bath.ac.uk/; www. ton.cvm.tamu.edu/lab/ www. ton.html; www.
  • Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
  • part or all of the non human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties.
  • humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Prestaet al., J. Immunol.
  • the anti-TNF antibody can also be optionally generated by immunization of a transgenic animal (e.g., mouse, rat, hamster, non-human primate, and the like) capable of producing a repertoire of human antibodies, as described herein and/or as known in the art.
  • a transgenic animal e.g., mouse, rat, hamster, non-human primate, and the like
  • Cells that produce a human anti-TNF antibody can be isolated from such animals and immortalized using suitable methods, such as the methods described herein.
  • Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be produced by known methods (e.g., but not limited to, U.S. Pat.
  • mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement.
  • the endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by endogenous genes.
  • peptide display libraries Screening antibodies for specific binding to similar proteins or fragments can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure antibody screening of peptide display libraries is well known in the art.
  • the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long.
  • bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence.
  • Antibodies of the present invention can also be prepared using at least one anti- TNF antibody encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk. Such animals can be provided using known methods. See, e.g., but not limited to, US patent nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of which is entirely incorporated herein by reference.
  • Antibodies of the present invention can additionally be prepared using at least one anti-TNF antibody encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured therefrom.
  • transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95- 118 (1999) and references cited therein.
  • transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources.
  • antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv’s), including tobacco seeds and potato tubers.
  • scFv single chain antibodies
  • the antibodies of the invention can bind human TNF with a wide range of affinities (KD).
  • KD affinities
  • at least one human mAb of the present invention can optionally bind human TNF with high affinity.
  • a human mAb can bind human TNF with a KD equal to or less than about 10 -7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10 -7 , 10 -8 , 10 -9 ,10 -10 , 10 -11 , 10 -12 , 10- 13 or any range or value therein.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method.
  • any suitable method See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed.,
  • affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH).
  • affinity and other antigen-binding parameters e.g., KD, K a , Kd
  • KD, K a , Kd are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, such as the buffer described herein.
  • nucleic Acid Molecules Using the information provided herein, such as the nucleotide sequences encoding at least 70-100% of the contiguous amino acids of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, specified fragments, variants or consensus sequences thereof, or a deposited vector comprising at least one of these sequences, a nucleic acid molecule of the present invention encoding at least one anti- TNF antibody comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2 and 3 and/or all of the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 can be obtained using methods described herein or as known in the art.
  • Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof.
  • the DNA can be triple -stranded, double- stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS: 1-3) or light chain (e.g., SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequence for an anti-TNF antibody or variable region (e.g., SEQ ID NOS:7,8); and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one anti-TNF antibody as described herein and/or as known in the art.
  • ORF open reading frame
  • introns e.g., but not limited to, at least one specified portion of at least one CDR, as CDR1, CDR2 and
  • nucleic acid variants that code for specific anti-TNF antibodies of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.
  • isolated nucleic acid molecules of the present invention include SEQ ID NOS: 10, 11, 12, 13,
  • HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HC variable region and LC variable region respectively, HC CDR1, HC CDR2, HC CDR3, HC variable region and LC variable region.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding an anti-TNF antibody can include, but are not limited to, those encoding the amino acid sequence of an antibody fragment, by itself; the coding sequence for the entire antibody or a portion thereof; the coding sequence for an antibody, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5’ and 3’ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities.
  • the sequence encoding an antibody can be fused to a marker sequence, such as a
  • the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein.
  • the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
  • the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences.
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
  • polynucleotides of this invention will encode at least a portion of an antibody encoded by the polynucleotides described herein.
  • the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an antibody of the present invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference. Construction of Nucleic Acids.
  • the isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well-known in the art.
  • the nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa- histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the coding sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.
  • Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra or Sambrook, supra).
  • RNA, cDNA, genomic DNA, or any combination thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
  • the isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra ; or Sambrook, supra).
  • a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur.
  • the degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
  • the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%.
  • the degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis
  • PCR polymerase chain reaction
  • in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al.,
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • Chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.
  • the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
  • a nucleic acid sequence of the present invention for example a cDNA or a genomic sequence encoding an antibody of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell.
  • transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
  • isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in intron) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
  • endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.
  • the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one anti-TNF antibody by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating site at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
  • Expression vectors will preferably but optionally include at least one selectable marker.
  • markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat.Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (GS, US Pat.Nos. 5,122,464; 5,770,359; 5,827,739) resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E.
  • MTX methotrexate
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • coli and other bacteria or prokaryotes (the above patents are entirely incorporated hereby by reference).
  • Appropriate culture mediums and conditions for the above- described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16- 18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
  • At least one antibody of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an antibody to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to an antibody of the present invention to facilitate purification. Such regions can be removed prior to final preparation of an antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.
  • nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an antibody of the present invention.
  • Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
  • mammalian cells useful for the production of the antibodies, specified portions or variants thereof, are mammalian cells.
  • Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used.
  • COS-1 e.g., ATCC CRL 1650
  • COS-7 e.g., ATCC CRL-1651
  • HEK293, BHK21 e.g., ATCC CRL-10
  • CHO e.g., ATCC CRL 1610
  • BSC-1 e.g., ATCC CRL-26 cell lines
  • Cos-7 cells CHO cells
  • hep G2 cells hep G2 cells
  • HeLa cells and the like which are readily available from, for example, American Type Culture Collection, Manassas, VA (www. atcc.org).
  • Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells.
  • Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851).
  • the recombinant cell is a P3X63Ab8.653 or a SP2/0-Agl4 cell.
  • Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat.Nos. 5,168,062; 5,385,839), an HSV tk promoter, apgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (US Pat.No.
  • a promoter e.g., late or early SV40 promoters, the CMV promoter (US Pat.Nos. 5,168,062; 5,385,839)
  • HSV tk promoter e.g., the CMV promoter (US Pat.Nos. 5,168,062; 5,385,839)
  • HSV tk promoter e.g., the CMV promoter (US Pat.Nos. 5,168,062; 5,385,839)
  • HSV tk promoter e.g.,
  • At least one human immunoglobulin promoter at least one human immunoglobulin promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • an enhancer, and/or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • polyadenylation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
  • gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.
  • An anti-TNF antibody can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification.
  • HPLC high performance liquid chromatography
  • Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
  • a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells.
  • the antibody of the present invention can be glycosylated or can be non- glycosylated, with glycosylated preferred.
  • Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.
  • the isolated antibodies of the present invention comprising all of the heavy chain variable CDR regions of SEQ ID NOS: 1, 2 and 3 and/or all of the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6, comprise antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody.
  • the human antibody or antigen-binding fragment binds human TNF and, thereby partially or substantially neutralizes at least one biological activity of the protein.
  • An antibody, or specified portion or variant thereof, that partially or preferably substantially neutralizes at least one biological activity of at least one TNF protein or fragment can bind the protein or fragment and thereby inhibit activities mediated through the binding of TNF to the TNF receptor or through other TNF-dependent or mediated mechanisms.
  • neutralizing antibody refers to an antibody that can inhibit an TNF-dependent activity by about 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay.
  • the capacity of an anti-TNF antibody to inhibit an TNF- dependent activity is preferably assessed by at least one suitable TNF protein or receptor assay, as described herein and/or as known in the art.
  • a human antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
  • the human antibody comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
  • Antibodies of this type can be prepared by employing a transgenic mouse or other transgenic non-human mammal comprising at least one human light chain (e.g., IgG,
  • the anti-human TNF human antibody comprises an IgGl heavy chain and a IgGl light chain.
  • an antibody or “antibodies”, include biosimilar antibody molecules approved under the Biologies Price Competition and Innovation Act of 2009 (BPCI Act) and similar laws and regulations globally. Under the BPCI Act, an antibody may be demonstrated to be biosimilar if data show that it is “highly similar” to the reference product notwithstanding minor differences in clinically inactive components and are "expected” to produce the same clinical result as the reference product in terms of safety, purity and potency ( Endocrine Practice : February 2018, Vol. 24, No. 2, pp. 195- 204). These biosimilar antibody molecules are provided an abbreviated approval pathway, whereby the applicant relies upon the innovator reference product's clinical data to secure regulatory approval.
  • SIMPONI® is the original innovator reference anti-TNF antibody that was FDA approved based on successful clinical trials. Golimumab has been on sale in the United States since 2009.
  • the TNF inhibitor comprises the anti-TNF antibody SIMPONI® (golimumab), or an antigen-binding fragment thereof comprising the sequences shown below.
  • SIMPONI® anti-TNF antibody SIMPONI®
  • an antigen-binding fragment thereof comprising the sequences shown below.
  • Example anti-TNF antibody sequences e.g., SIMPONI® (golimumab)
  • Heavy chain CDRs HCDRs
  • light chain CDRs LCDRs
  • Rabat Amino acid sequence of golimumab heavy chain (HC) with CDRs underlined: (SEQ ID NO:36
  • VH variable heavy chain
  • VL variable light chain
  • HCDR1 golimumab heavy chain complementarity determining region 1
  • HCDR2 golimumab antibody heavy chain complementarity determining region 2
  • HCDR3 amino acid sequence of golimumab heavy chain complementarity determining region 3 (HCDR3): (SEQ ID NO:42)
  • LCDR1 golimumab light chain complementarity determining region 1
  • LCDR2 golimumab light chain complementarity determining region 2
  • At least one antibody of the invention binds at least one specified epitope specific to at least one TNF protein, subunit, fragment, portion or any combination thereof.
  • the at least one epitope can comprise at least one antibody binding region that comprises at least one portion of said protein, which epitope is preferably comprised of at least one extracellular, soluble, hydrophilic, external or cytoplasmic portion of said protein.
  • the at least one specified epitope can comprise any combination of at least one amino acid sequence of at least 1-3 amino acids to the entire specified portion of contiguous amino acids of the SEQ ID NO: 9.
  • the human antibody or antigen-binding fragment of the present invention will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one light chain variable region.
  • the antibody or antigen-binding portion or variant can comprise at least one of the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3, and/or a light chain CDR3 having the amino acid sequence of SEQ ID NO:6.
  • the antibody or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 1, 2, and/or 3).
  • the antibody or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 4, 5, and/or 6).
  • the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen binding fragment have the amino acid sequence of the corresponding CDR of at least one ofmAb TNV148, TNV14, TNV15, TNV196, TNV118, TNV32, TNV86, as described herein.
  • Such antibodies can be prepared by chemically joining together the various portions (e.g., CDRs, framework) of the antibody using conventional techniques, by preparing and expressing a (i.e., one or more) nucleic acid molecule that encodes the antibody using conventional techniques of recombinant DNA technology or by using any other suitable method.
  • the anti-TNF antibody can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence.
  • the anti-TNF antibody comprises at least one of heavy chain variable region, optionally having the amino acid sequence of SEQ ID NO:7 and/or at least one light chain variable region, optionally having the amino acid sequence of SEQ ID NO:8.
  • antibodies that bind to human TNF and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al.,IntJMol. Med, 1 (A) : 863 -868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein.
  • a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus that can undergo functional rearrangement, can be immunized with human TNF or a fragment thereof to elicit the production of antibodies.
  • the antibody producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and/or as known in the art.
  • the antibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
  • the invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
  • antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can bind human TNF with high affinity (e.g., KD less than or equal to about 10 9 M).
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g, charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • amino acids that make up anti-TNF antibodies of the present invention are often abbreviated.
  • the amino acid designations can be indicated by designating the amino acid by its single letter code, its three-letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994):
  • An anti-TNF antibody of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • amino acid substitutions a skilled artisan would make depends on many factors, including those described above.
  • the number of amino acid substitutions, insertions or deletions for any given anti-TNF antibody, fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein.
  • Amino acids in an anti-TNF antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)).
  • the latter procedure introduces single alanine mutations at every residue in the molecule.
  • the resulting mutant molecules are then tested for biological activity, such as, but not limited to at least one TNF neutralizing activity.
  • Sites that are critical for antibody binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffmity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
  • Anti-TNF antibodies of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from 1 to all of the contiguous amino acids of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6.
  • A(n) anti-TNF antibody can further optionally comprise a polypeptide of at least one of 70-100% of the contiguous amino acids of at least one of SEQ ID NOS:7,
  • amino acid sequence of an immunoglobulin chain, or portion thereof has about 70-100% identity (e.g., 70, 71,
  • amino acid sequence of a light chain variable region can be compared with the sequence of SEQ ID NO: 8, or the amino acid sequence of a heavy chain CDR3 can be compared with SEQ ID NO:7.
  • 70-100% amino acid identity i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein
  • a suitable computer algorithm as known in the art.
  • the antibodies of the present invention can comprise any number of contiguous amino acid residues from an antibody of the present invention, wherein that number is selected from the group of integers consisting of from 10-100% of the number of contiguous residues in an anti-TNF antibody.
  • this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein.
  • the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as at least 2, 3, 4, or 5.
  • the present invention includes at least one biologically active antibody of the present invention.
  • Biologically active antibodies have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non synthetic), endogenous or related and known antibody. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity, are well known to those of skill in the art.
  • the invention relates to human antibodies and antigen-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety.
  • modification can produce an antibody or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half- life).
  • the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
  • the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • a polyalkane glycol e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)
  • carbohydrate polymer e.g., amino acid polymer or polyvinyl pyrolidone
  • the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • the modified antibodies and antigen-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody.
  • Each organic moiety that is bonded to an antibody or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
  • fatty acid encompasses mono-carboxylic acids and di -carboxylic acids.
  • Hydrophilic polymers suitable for modifying antibodies of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
  • polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
  • carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
  • polymers of hydrophilic amino acids e.g., polylysine,
  • the hydrophilic polymer that modifies the antibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
  • a molecular weight of about 800 to about 150,000 Daltons for example, PEG5000 and PEG20.000. wherein the subscript is the average molecular weight of the polymer in Daltons, can be used.
  • the hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods.
  • a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
  • an activated carboxylate e.g., activated with N, N-carbonyl diimidazole
  • Fatty acids and fatty acid esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaturation.
  • Fatty acids that are suitable for modifying antibodies of the invention include, for example, n- dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (Cis, stearate), n-eicosanoate (C20, arachidate) , n-docosanoate (C22, behenate), n- triacontanoate (C30), n-tetracontanoate (C40), ci-D9-octadecanoate (Cis, oleate), all cis- D5.8.1 1.14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecane
  • modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents.
  • An "activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
  • amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.
  • Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like.
  • An aldehyde functional group can be coupled to amine- or hydrazide- containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
  • Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
  • An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
  • Suitable linker moieties include, for example, tetraethylene glycol, - (CH 2 ) 3 -, -NH-(CH 2 ) 6 -NH-, -(CH 2 ) 2 -NH- and -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH- NH-.
  • Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc- diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.
  • a mono-Boc-alkyldiamine e.g., mono-Boc-ethylenediamine, mono-Boc- diaminohexane
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid.
  • TFA trifluoroacetic acid
  • the modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent.
  • a modifying agent for example, the organic moieties can be bonded to the antibody in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
  • Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or antigen binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention.
  • Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an antibody of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al. , Bioorg.
  • suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al. , Bioorg.
  • Anti-Idiotype Antibodies to Anti-TNF Antibody Compositions In addition to monoclonal or chimeric anti-TNF antibodies, the present invention is also directed to an anti-idiotypic (anti-id) antibody specific for such antibodies of the invention.
  • An anti-id antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding region of another antibody.
  • the anti-id can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Id antibody with the antibody or a CDR containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-id antibody.
  • the anti-id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the present invention also provides at least one anti-TNF antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more anti-TNF antibodies thereof, as described herein and/or as known in the art that are provided in a non-naturally occurring composition, mixture or form.
  • Such compositions comprise non-naturally occurring compositions comprising at least one or two full length, C- and/or N- terminally deleted variants, domains, fragments, or specified variants, of the anti-TNF antibody amino acid sequence selected from the group consisting of 70-100% of the contiguous amino acids of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, or specified fragments, domains or variants thereof.
  • Preferred anti-TNF antibody compositions include at least one or two full length, fragments, domains or variants as at least one CDR or LBR containing portions of the anti-TNF antibody sequence of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or specified fragments, domains or variants thereof. Further preferred compositions comprise 40-99% of at least one of 70-100% of SEQ ID NOS:l, 2, 3, 4,
  • composition percentages are by weight, volume, concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein.
  • Anti-TNF antibody compositions of the present invention can further comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local an
  • Non-limiting examples of such cytokines include, but are not limted to, any of IL-1 to IL-23.
  • Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
  • Such anti -cancer or anti-infectives can also include toxin molecules that are associated, bound, co-formulated or co-administered with at least one antibody of the present invention.
  • the toxin can optionally act to selectively kill the pathologic cell or tissue.
  • the pathologic cell can be a cancer or other cell.
  • Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, diphtheria toxin, a venom toxin, or a bacterial toxin.
  • toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or viruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death.
  • toxins may include, but are not limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin- 1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like.
  • Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella hoydii, and Shigella sonnei), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens
  • Anti-TNF antibody compounds, compositions or combinations of the present invention can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • suitable auxiliaries are preferred.
  • Non limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington ’s Pharmaceutical Sciences,
  • Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the anti-TNF antibody, fragment or variant composition as well known in the art or as described herein.
  • compositions include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • One preferred amino acid is glycine.
  • Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffmose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
  • Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffmose.
  • Anti-TNF antibody compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • Preferred buffers for use in the present compositions are organic acid salts such as citrate.
  • anti-TNF antibody compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
  • polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycols,
  • compositions according to the invention are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy”, 19 th ed., Williams & Williams, (1995), and in the “Physician’s Desk Reference”, 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by reference.
  • Preferred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.
  • the invention provides for stable formulations, which is preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one anti- TNF antibody in a pharmaceutically acceptable formulation.
  • Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent.
  • Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4.
  • Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3.
  • benzyl alcohol e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.
  • alkylparaben(s) e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%),
  • the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one anti-TNF antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
  • the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one anti-TNF antibody, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one anti-TNF antibody in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
  • the at least one anti-TNF antibody used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.
  • the range of at least one anti-TNF antibody in the product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 pg/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
  • the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
  • preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.
  • concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
  • excipients e.g. isotonicity agents, buffers, antioxidants, preservative enhancers
  • An isotonicity agent such as glycerin, is commonly used at known concentrations.
  • a physiologically tolerated buffer is preferably added to provide improved pH control.
  • the formulations can cover a wide range of pH, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0.
  • the formulations of the present invention have pH between about 6.8 and about 7.8.
  • Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
  • additives such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic® (polyols) F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® (polyols), other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation.
  • a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene
  • the formulations of the present invention can be prepared by a process which comprises mixing at least one anti-TNF antibody and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent.
  • a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous
  • a measured amount of at least one anti- TNF antibody in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations.
  • Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
  • the claimed formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-TNF antibody that is reconstituted with a second vial containing water, a preservative and/or excipient, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
  • a preservative and/or excipient preferably a phosphate buffer and/or saline and a chosen salt
  • Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40°C and retain the biologically activity of the protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years.
  • the solutions of at least one anti-TNF antibody in the invention can be prepared by a process that comprises mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one antibody in water or buffer is combined in quantities sufficient to provide the protein and optionally a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
  • the claimed products can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-TNF antibody that is reconstituted with a second vial containing the aqueous diluent.
  • a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
  • the claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one anti-TNF antibody that is reconstituted with a second vial containing the aqueous diluent.
  • the clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the at least one antibody solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients.
  • Recognized devices comprising these single vial systems include those pen- injector devices for delivery of a solution such as B-D ® (pen injector device), NOVOPEN ® (pen injector device), AUTOPEN ® (pen injector device), OPTIPEN ® (pen injector device), GENOTROPIN PEN ® (pen injector device), -HUMATROPEN ® (pen injector device), BIOJECTOR ® (pen injector device), Reco-Pen, Humaject, J-tip Needle-Free Injector, Intraject, Medi-Ject, e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.
  • Recognized devices comprising a dual vial system include those pen-injector systems for reconstituting a lyophilized drug in a cartridge for delivery of the reconstituted solution such as the HUMATROPEN ® (pen injector device).
  • the products presently claimed include packaging material.
  • the packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.
  • the packaging material of the present invention provides instructions to the patient to reconstitute the at least one anti-TNF antibody in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product.
  • the label indicates that such solution can be used over a period of 2-24 hours or greater.
  • the presently claimed products are useful for human pharmaceutical product use.
  • the formulations of the present invention can be prepared by a process that comprises mixing at least one anti-TNF antibody and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt. Mixing the at least one antibody and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one antibody in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
  • the claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-TNF antibody that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent.
  • a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
  • At least one anti-TNF antibody in either the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
  • the present invention also provides a method for modulating or treating at least one TNF related disease, in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one dual integrin antibody of the present invention.
  • the present invention also provides a method for modulating or treating at least one TNF related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of obesity, an immune related disease, a cardiovascular disease, an infectious disease, a malignant disease, a neurologic disease, or Type 1 Diabetes (T1D) and beta-cell dysfunction.
  • T1D Type 1 Diabetes
  • the present invention also provides a method for modulating or treating at least one immune related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile , systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener’s granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplants,
  • the present invention also provides a method for modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic arteriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post perfusion syndrome, cardiopulmonary bypass inflammation response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrythmias, ventricular fibrillation, His bundle arrythmias, atrioventricular block, bundle branch block
  • Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • the present invention also provides a method for modulating or treating at least one infectious disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (A,B or C, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, e.
  • coli 0157:h7 hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellular, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epididymitis, legionella, Lyme disease, influenza a, Epstein Barr virus, viral-associated hemophagocytic syndrome, vital encephalitis/aseptic meningitis, and the like.
  • the present invention also provides a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or LAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adeno
  • the present invention also provides a method for modulating or treating at least one neurologic disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders' such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug- induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranuclear Palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Dr
  • Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti- TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Such a method can optionally further comprise co-administration or combination therapy for treating such immune diseases, wherein the administering of said at least one anti-TNF antibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant
  • Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
  • TNF antagonists suitable for compositions, combination therapy, co administration, devices and/or methods of the present invention include, but are not limited to, anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules which bind specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signaling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF activity, such as angiotensin converting enzyme (ACE) inhibitors
  • MAP mitogen activated protein
  • ACE angiotensin converting enzyme
  • a "tumor necrosis factor antibody,” “TNF antibody,” “TNFa antibody,” or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNFa activity in vitro, in situ and/or preferably in vivo.
  • a suitable TNF human antibody of the present invention can bind TNFa and includes anti-TNF antibodies, antigen-binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNFa.
  • a suitable TNF antibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
  • Chimeric antibody cA2 consists of the antigen binding variable region of the high-affinity neutralizing mouse anti -human TNFa IgGl antibody, designated A2, and the constant regions of a human IgGl, kappa immunoglobulin.
  • the human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half-life and decreases the immunogenicity of the antibody.
  • the avidity and epitope specificity of the chimeric antibody cA2 is derived from the variable region of the murine antibody A2.
  • a preferred source for nucleic acids encoding the variable region of the murine antibody A2 is the A2 hybridoma cell line.
  • Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant human TNFa in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNFa, the affinity constant of chimeric antibody cA2 was calculated to be 1.04X10M -1 .
  • murine monoclonal antibody A2 is produced by a cell line designated cl34A.
  • Chimeric antibody cA2 is produced by a cell line designated cl68A.
  • TNF Receptor Molecules Preferred TNF receptor molecules useful in the present invention are those that bind TNFa with high affinity (see, e.g., Feldmann et al., International Publication No. WO 92/07076 (published April 30, 1992); Schall et al., Cell 61: 361-370 (1990); and Loetscher et al., Cell 61: 351-359 (1990), which references are entirely incorporated herein by reference) and optionally possess low immunogenicity.
  • the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention.
  • Truncated forms of these receptors comprising the extracellular domains (ECD) of the receptors or functional portions thereof (see, e.g., Corcoran et al., Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present invention.
  • Truncated forms of the TNF receptors, comprising the ECD have been detected in urine and serum as 30 kDa and 40 kDa TNFa inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem. 265: 1531-1536 (1990)).
  • TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules which are useful in the methods and compositions of the present invention.
  • the TNF receptor molecules which can be used in the invention are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, can contribute to the therapeutic results achieved.
  • TNF receptor multimeric molecules useful in the present invention comprise all or a functional portion of the ECD of two or more TNF receptors linked via one or more polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG).
  • the multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule.
  • TNF immunoreceptor fusion molecules useful in the methods and compositions of the present invention comprise at least one portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers, or hetero- or homo- multimers. The immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusion protein. TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al., Eur. J. Immunol.
  • a functional equivalent, derivative, fragment or region of TNF receptor molecule refers to the portion of the TNF receptor molecule, or the portion of the TNF receptor molecule sequence which encodes TNF receptor molecule, that is of sufficient size and sequences to functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNFD with high affinity and possess low immunogenicity).
  • a functional equivalent of TNF receptor molecule also includes modified TNF receptor molecules that functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNFa with high affinity and possess low immunogenicity).
  • a functional equivalent of TNF receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid).
  • SILENT substitution of one acidic amino acid for another acidic amino acid
  • substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid.
  • Cytokines include any known cytokine. See, e.g., CopewithCytokines.com. Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof.
  • Any method of the present invention can comprise a method for treating a TNF mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one anti- TNF antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Such a method can optionally further comprise co-administration or combination therapy for treating such immune diseases, wherein the administering of said at least one anti-TNF antibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker,
  • treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one anti-TNF antibody composition that total, on average, a range from at least about 0.01 to 500 milligrams of at least one anti-TNF antibody per kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams antibody /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
  • the effective serum concentration can comprise 0.1-5000 pg/ml serum concentration per single or multiple administration.
  • Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e.. repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
  • Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
  • 98, 99 and/or 100-500 mg/kg/administration or any range, value or fraction thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9,
  • the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
  • 0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
  • treatment of humans or animals can be provided as a one-time or periodic dosage of at least one antibody of the present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container.
  • the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.
  • the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
  • the formulation is sterilized by known or suitable techniques.
  • Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • TNF antibodies of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
  • Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.
  • Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aqueous solution or a sterile injectable solution or suspension in a solvent.
  • As the usable vehicle or solvent water, Ringer's solution, isotonic saline, etc.
  • sterile involatile oil can be used as an ordinary solvent, or suspending solvent.
  • any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono- or di- or tri -glycerides.
  • Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.
  • the invention further relates to the administration of at least one anti-TNF antibody by parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.
  • At least one anti-TNF antibody composition can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as, but not limited to, creams and suppositories; for buccal, or sublingual administration such as, but not limited to, in the form of tablets or capsules; or intranasally such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al.
  • parenteral subcutaneous, intramuscular or intravenous
  • vaginal or rectal administration particularly in semisolid forms such as, but not limited to, creams and suppositories
  • At least one anti-TNF antibody composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
  • at least one anti-TNF antibody can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of antibodies are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of antibody in an aerosol.
  • Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles.
  • Metered dose inhalers like the VENTOLIN ® (metered dose inhaler), typically use a propellant gas and require actuation during inspiration (See, e g., WO 94/16970, WO 98/35888).
  • Dry powder inhalers like Turbuhaler (Astra), Rotahaler (Glaxo), DISKUS ® (inhaler) (Glaxo), SPIROS ® (inhaler) (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference).
  • Nebulizers like AERX ® (nebulizer) Aradigm, the ULTRA VENT ® (nebulizer) (Mallinckrodt), and the Acorn II nebulizer (Marquest Medical Products)
  • a composition comprising at least one anti-TNF antibody is delivered by a dry powder inhaler or a sprayer.
  • an inhalation device for administering at least one antibody of the present invention is advantageously reliable, reproducible, and accurate.
  • the inhalation device can optionally deliver small dry particles, e.g. less than about 10 pm, preferably about 1-5 pm, for good respirability.
  • a spray including TNF antibody composition protein can be produced by forcing a suspension or solution of at least one anti -TNF antibody through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
  • particles of at least one anti-TNF antibody composition protein delivered by a sprayer have a particle size less than about 10 pm, preferably in the range of about 1 pm to about 5 pm, and most preferably about 2 pm to about 3 pm.
  • Formulations of at least one anti-TNF antibody composition protein suitable for use with a sprayer typically include antibody composition protein in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one anti-TNF antibody composition protein per ml of solution or mg/gm, or any range or value therein, e.g., but not limited to, .1, .2., .3, .4, .5, .6, .7, .8, .9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating antibody composition proteins include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the antibody composition protein formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the antibody composition protein caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as TNF antibodies, or specified portions or variants, can also be included in the formulation.
  • Antibody composition protein can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
  • a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
  • a compressed air source is used to create a high-velocity air jet through an orifice.
  • a low-pressure region is created, which draws a solution of antibody composition protein through a capillary tube connected to a liquid reservoir.
  • the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
  • a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
  • an ultrasonic nebulizer high-frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the formulation of antibody composition protein either directly or through a coupling fluid, creating an aerosol including the antibody composition protein.
  • particles of antibody composition protein delivered by a nebulizer have a particle size less than about 10 pm, preferably in the range of about 1 pm to about 5 pm, and most preferably about 2 pm to about 3 pm.
  • Formulations of at least one anti-TNF antibody suitable for use with a nebulizer, either jet or ultrasonic typically include a concentration of about 0.1 mg to about 100 mg of at least one anti-TNF antibody protein per ml of solution.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the at least one anti-TNF antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating at least one anti-TNF antibody composition proteins include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating at least one anti- TNF antibody include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the at least one anti-TNF antibody formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one anti-TNF antibody caused by atomization of the solution in forming an aerosol.
  • a surfactant which can reduce or prevent surface-induced aggregation of the at least one anti-TNF antibody caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation.
  • Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a
  • a propellant, at least one anti-TNF antibody, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 pm, preferably about 1 pm to about 5 pm, and most preferably about 2 pm to about 3 pm.
  • the desired aerosol particle size can be obtained by employing a formulation of antibody composition protein produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like.
  • Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
  • Formulations of at least one anti-TNF antibody for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one anti-TNF antibody as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
  • the propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.
  • the propellant is a hydrofluorocarbon.
  • the surfactant can be chosen to stabilize the at least one anti-TNF antibody as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
  • Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases, solution aerosols are preferred using solvents such as ethanol. Additional agents known in the art for formulation of a protein can also be included in the formulation.
  • Formulations for oral rely on the co administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation.
  • adjuvants e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether
  • enzymatic inhibitors e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol
  • the active constituent compound of the solid-type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffmose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
  • at least one additive including sucrose, lactose, cellulose, mannitol, trehalose, raffmose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
  • These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha. -tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
  • additives e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha. -tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
  • Tablets and pills can be further processed into enteric-coated preparations.
  • the liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations allowable for medical use. These preparations can contain inactive diluting agents ordinarily used in said field, e.g., water.
  • Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673).
  • carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents orally are known in the art.
  • compositions and methods of administering at least one anti-TNF antibody include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670).
  • Mucous surfaces suitable for application of the emulsions of the present invention can include comeal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration.
  • Formulations for vaginal or rectal administration can contain as excipients, for example, polyalkyleneglycols, Vaseline, cocoa butter, and the like.
  • Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. Pat. Nos. 5,849,695).
  • the at least one anti-TNF antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
  • a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
  • suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. Nos.
  • a dosage form can contain a pharmaceutically acceptable non-toxic salt of the compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-dibenz
  • the compounds of the present invention or, preferably, a relatively insoluble salt such as those just described can be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
  • Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
  • Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non- antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919.
  • the compounds or, preferably, relatively insoluble salts such as those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals.
  • Additional slow release, depot or implant formulations, e.g. gas or liquid liposomes are known in the literature (U.S. Pat. Nos. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).
  • Example 1 Cloning and Expression of TNF antibody in Mammalian Cells.
  • a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the antibody coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
  • LTRS long terminal repeats
  • CMV cytomegalovirus
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hy
  • Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded antibody.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio/Technology 10: 169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of antibodies.
  • the expression vectors pCl and pC4 contain the strong promoter (UTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
  • the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • Plasmid pC4 is used for the expression of TNF antibody.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM, Fife Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
  • MTX methotrexate
  • a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated into one or more chromosome(s) of the host cell.
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • high efficiency promoters can also be used for the expression, e.g., the human beta-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the TNF in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB101 or XL-1 Blue cells are then transformed, and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • 5 pig of the expression plasmid pC4 is cotransfected with 0.5 pig of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 pig /ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 pig /ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate ( 1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
  • Example 2 Generation of High Affinity Human IgG Monoclonal Antibodies Reactive with Human TNF Using Transgenic Mice.
  • mice have been used that contain human heavy and light chain immunoglobulin genes to generate high affinity, completely human, monoclonal antibodies that can be used therapeutically to inhibit the action of TNF for the treatment of one or more TNF-mediated disease.
  • CBA/J x C57/BL6/J F2 hybrid mice containing human variable and constant region antibody transgenes for both heavy and light chains are immunized with human recombinant TNF (Taylor et al., Inti. Immunol. 6:579-591 (1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M., Nature Biotech.
  • BSA bovine serum albumin
  • CO2 carbon dioxide
  • DMSO dimethyl sulfoxide
  • EIA enzyme immunoassay
  • FBS fetal bovine serum
  • H2O2 hydrogen peroxide
  • HRP horseradish peroxidase
  • ID - intradermal Ig - immunoglobulin
  • TNF tissue necrosis factor alpha
  • IP intraperitoneal
  • IV intravenous
  • Mab monoclonal antibody
  • OD optical density
  • OPD - o- Phenylenediamine dihydrochloride
  • PEG polyethylene glycol
  • PSA penicillin, streptomycin, amphotericin
  • SQ subcutaneous
  • v/v - volume per volume w/v - weight per volume.
  • Transgenic mice that can express human antibodies are known in the art (and are commercially available (e.g., from GenPharm International, San Jose, CA; Abgenix, Freemont, CA, and others) that express human immunoglobulins but not mouse IgM or IgK.
  • transgenic mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins (Lonberg, et al., Nature 368:856-859 (1994)).
  • the light chain transgene can be derived, e.g., in part from a yeast artificial chromosome clone that includes nearly half of the germline human VK region.
  • the heavy-chain transgene can encode both human m and human yl (Fishwild. et al., Nature Biotechnology 14:845-851 (1996)) and/or g3 constant regions.
  • Mice derived from appropriate genotypic lineages can be used in the immunization and fusion processes to generate fully human monoclonal antibodies to TNF.
  • Immunization One or more immunization schedules can be used to generate the anti-TNF human hybridomas. The first several fusions can be performed after the following exemplary immunization protocol, but other similar known protocols can be used.
  • mice are immunized IP and/or ID with 1-1000 mg of recombinant human TNF emulsified with an equal volume of TITERMAX or complete Freund's adjuvant in a final volume of 100-400pL (e.g., 200).
  • Each mouse can also optionally receive 1-10 pg in 100 pL physiological saline at each of 2 SQ sites.
  • the mice can then be immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400 pg) and SQ (1-400 pg x 2) with TNF emulsified with an equal volume of TITERMAX or incomplete Freund's adjuvant.
  • mice can be bled 12-25 and 25-40 days later by retro-orbital puncture without anti-coagulant.
  • the blood is then allowed to clot at RT for one hour and the serum is collected and tittered using an TNF EIA assay according to known methods. Fusions are performed when repeated injections do not cause titers to increase. At that time, the mice can be given a final IV booster injection of 1-400 pg TNF diluted in 100 pL physiological saline.
  • mice can be euthanized by cervical dislocation and the spleens removed aseptically and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (PSA).
  • PBS cold phosphate buffered saline
  • PSA amphotericin B
  • the splenocytes are harvested by sterilely perfusing the spleen with PSA-PBS.
  • the cells are washed once in cold PSA-PBS, counted using Trypan blue dye exclusion and resuspended in RPMI 1640 media containing 25 mM Hepes.
  • Fusion can be carried out at a 1: 1 to 1: 10 ratio of murine myeloma cells to viable spleen cells according to known methods, e.g., as known in the art.
  • spleen cells and myeloma cells can be pelleted together. The pellet can then be slowly resuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight 1,450, Sigma) at 37 ⁇ C. The fusion can then be stopped by slowly adding 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37 ⁇ C) over 1 minute.
  • the fused cells are centrifuged for 5 minutes at 500- 1500 rpm.
  • the cells are then resuspended in HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 mg/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 10% 653 -conditioned RPMI 1640/Hepes media, 50 mM 2-mercaptoethanol, 100 mM hypoxanthine, 0.4 mM aminopterin, and 16 mM thymidine) and then plated at 200 mL/well in fifteen 96-well flat bottom tissue culture plates. The plates are then placed in a humidified 37 ⁇ C incubator containing 5% CC 2 and 95% air for 7-10 days.
  • HAT medium RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (H
  • Solid phase EIA can be used to screen mouse sera for human IgG antibodies specific for human TNF. Briefly, plates can be coated with TNF at 2 mg/mL in PBS overnight. After washing in 0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v) BSA in PBS, 200 pL/well for 1 hour at RT. Plates are used immediately or frozen at -20 ⁇ C for future use. Mouse serum dilutions are incubated on the TNF coated plates at 50 pL/well at RT for 1 hour.
  • the plates are washed and then probed with 50 pL/well HRP-labeled goat anti-human IgG, Fc specific diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT.
  • the plates can again be washed and 100 pL/well of the citrate-phosphate substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H2O2 and 1 mg/mL OPD) is added for 15 minutes at RT. Stop solution (4N sulfuric acid) is then added at 25 pL/well and the OD's are read at 490 nm via an automated plate spectrophotometer.
  • Hybridomas as above, can be simultaneously assayed for reactivity to TNF using a suitable RIA or other assay. For example, supernatants are incubated on goat anti-human IgG Fc plates as above, washed and then probed with radiolabeled TNF with appropriate counts per well for 1 hour at RT. The wells are washed twice with PBS and bound radiolabeled TNF is quantitated using a suitable counter.
  • Human IgGlK anti-TNF secreting hybridomas can be expanded in cell culture and serially subcloned by limiting dilution.
  • the resulting clonal populations can be expanded and cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen.
  • Isotyping Isotype determination of the antibodies can be accomplished using an EIA in a format similar to that used to screen the mouse immune sera for specific titers.
  • TNF can be coated on 96- well plates as described above and purified antibody at 2 mg/mL can be incubated on the plate for one hour at RT. The plate is washed and probed with HRP labeled goat anti-human IgGi or HRP labeled goat anti-human IgG3 diluted at 1:4000 in 1% BSA-PBS for one hour at RT. The plate is again washed and incubated with substrate solution as described above.
  • Binding Kinetics of Human Anti-Human TNF Antibodies with Human TNF Binding characteristics for antibodies can be suitably assessed using an TNF capture EIA and BIAcore technology, for example. Graded concentrations of purified human TNF antibodies can be assessed for binding to EIA plates coated with 2 mg/mL of TNF in assays as described above. The OD’s can be then presented as semi-log plots showing relative binding efficiencies.
  • Quantitative binding constants can be obtained, e.g., as follows, or by any other known suitable method.
  • a BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit.
  • HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cell of the chip at 5 pL/minute until a stable baseline is obtained.
  • N-ethyl-N’-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride in 200 pL water is added to 100 pL of a solution of 2.3 mg ofNHS (N-hydroxysuccinimide) in 200 pL water. Forty (40) pL of the resulting solution is injected onto the chip. Six pL of a solution of human TNF (15 mg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip, resulting in an increase of ca. 500 RU.
  • the buffer is changed to TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mM calcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 mg/mL BSA, pH 7.4) and flowed over the chip overnight to equilibrate it and to hydrolyze or cap any unreacted succinimide esters.
  • Antibodies are dissolved in the running buffer at 33.33, 16.67, 8.33, and 4.17 nM.
  • the flow rate is adjusted to 30 mL/min and the instrument temperature to 25°C.
  • Two flow cells are used for the kinetic runs, one on which TNF had been immobilized (sample) and a second, underivatized flow cell (blank).
  • 120 pL of each antibody concentration is injected over the flow cells at 30 pL/min (association phase) followed by an uninterrupted 360 seconds of buffer flow (dissociation phase).
  • the surface of the chip is regenerated (tissue necrosis factor alpha /antibody complex dissociated) by two sequential injections of 30 pL each of 2 M guanidine thiocyanate.
  • FIGS. 1-2 show the results of the relative binding efficiency of these antibodies. In this case, the avidity of the antibody for its cognate antigen (epitope) is measured. It should be noted that binding TNF directly to the EIA plate can cause denaturation of the protein and the apparent binding affinities cannot be reflective of binding to undenatured protein. Fifty percent binding is found over a range of concentrations.
  • Quantitative binding constants are obtained using BIAcore analysis of the human antibodies and reveals that several of the human monoclonal antibodies are very high affinity with KD in the range of 1x10 -9 to 7xl0 -12 .
  • Example 3 Generation of Human IgG Monoclonal Antibodies Reactive to Human TNFa.
  • BSA bovine serum albumin
  • CO2 carbon dioxide
  • DMSO dimethyl sulfoxide
  • EIA enzyme immunoassay
  • FBS fetal bovine serum
  • H2O2 hydrogen peroxide
  • HC heavy chain
  • HRP horseradish peroxidase
  • ID - intradermal Ig - immunoglobulin
  • TNF - tissue necrosis factor alpha IP - intraperitoneal
  • IV - intravenous
  • Mab monoclonal antibody
  • OD optical density
  • OPD - o- Phenylenediamine dihydrochloride
  • PEG polyethylene glycol
  • PSA penicillin, streptomycin, amphotericin
  • SQ subcutaneous
  • TNFa - tumor necrosis factor alpha v/v - volume per volume
  • w/v - weight per volume w/v - weight per volume.
  • Transgenic mice that contain human heavy and light chain immunoglobulin genes were utilized to generate totally human monoclonal antibodies that are specific to recombinant human TNFa. It is hoped that these unique antibodies can be used, as cA2 (Remicade) is used to therapeutically inhibit the inflammatory processes involved in TNFa-mediated disease with the benefit of increased serum half-life and decreased side effects relating to immunogenicity.
  • cA2 Remicade
  • mice that express human immunoglobulins have been developed by GenPharm International. These mice contain functional human antibody transgenes that undergo V(D)J joining, heavy -chain class switching and somatic mutation to generate a repertoire of antigen-specific human immunoglobulins (1).
  • the light chain transgenes are derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human VK locus.
  • the heavy-chain (HC) transgene encodes both human m and human g ⁇ (2) and/or g3 constant regions.
  • a mouse derived from the HCol2/KCo5 genotypic lineage was used in the immunization and fusion process to generate the monoclonal antibodies described here.
  • Human TNFa was purified from tissue culture supernatant from C237A cells by affinity chromatography using a column packed with the TNFa receptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4B (Pharmacia). The cell supernatant was mixed with one-ninth its volume of lOx Dulbecco’s PBS (D-PBS) and passed through the column at 4 °C at 4 mL/min. The column was then washed with PBS and the TNFa was eluted with 0.1 M sodium citrate, pH 3.5 and neutralized with 2 M Tris-HCl pH 8.5. The purified TNFa was buffer exchanged into 10 mM Tris, 0.12 M sodium chloride pH 7.5 and filtered through a 0.2 um syringe filter.
  • D-PBS lOx Dulbecco’s PBS
  • GenPharm mouse approximately 16 weeks old, was immunized IP (200 mL) and ID (100 mL at the base of the tail) with a total of 100 pg of TNFa (lot JG102298 or JG102098) emulsified with an equal volume of Titermax adjuvant on days 0, 12 and 28.
  • the mouse was bled on days 21 and 35 by retro-orbital puncture without anti-coagulant.
  • the blood was allowed to clot at RT for one hour and the serum was collected and tittered using TNFa solid phase EIA assay.
  • GenTNV was performed after the mouse was allowed to rest for seven weeks following injection on day 28.
  • the mouse with a specific human IgG titer of 1 : 160 against TNFa, was then given a final IV booster injection of 50 mg TNFa diluted in 100 mL physiological saline.
  • the mouse was euthanized by cervical dislocation and the spleen was removed aseptically and immersed in 10 mL of cold phosphate-buffered saline (PBS) containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (PSA).
  • PBS cold phosphate-buffered saline
  • PSA amphotericin B
  • the splenocytes were harvested by sterilely perfusing the spleen with PSA-PBS.
  • the cells were washed once in cold PSA-PBS, counted using a Coulter counter and resuspended in RPMI 1640 media containing 25 mM Hepes.
  • the non-secreting mouse myeloma fusion partner, 653 was received into Cell Biology Services (CBS) group on 5-14-97 from Centocor’s Product Development group.
  • CBS Cell Biology Services
  • the cell line was expanded in RPMI medium (JRH Biosciences) supplemented with 10% (v/v) FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine (all from JRH Biosciences) and cryopreserved in 95% FBS and 5% DMSO (Sigma), then stored in a vapor phase liquid nitrogen freezer in CBS.
  • the cell bank was sterile (Quality Control Centocor, Malvern) and free of mycoplasma (Bionique Laboratories). Cells were maintained in log phase culture until fusion. They were washed in PBS, counted, and viability determined (>95%) via trypan blue dye exclusion prior to fusion.
  • Human TNFa was produced by a recombinant cell line, named C237A, generated in Molecular Biology at Centocor.
  • the cell line was expanded in IMDM medium (JRH Biosciences) supplemented with 5% (v/v) FBS (Cell Culture Labs), 2 mM L-glutamine (all from JRH Biosciences), and 0.5 :g/mL mycophenolic acid, and cryopreserved in 95% FBS and 5% DMSO (Sigma), then stored in a vapor phase liquid nitrogen freezer in CBS (13).
  • the cell bank was sterile (Quality Control Centocor, Malvern) and free of mycoplasma (Bionique Laboratories).
  • the cell fusion was carried out using a 1: 1 ratio of 653 murine myeloma cells and viable murine spleen cells. Briefly, spleen cells and myeloma cells were pelleted together. The pellet was slowly resuspended over a 30 second period in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight of 1,450 g/mole, Sigma) at 37°C. The fusion was stopped by slowly adding 10.5 mL of RPMI media (no additives) (JRH) (37°C) over 1 minute. The fused cells were centrifuged for 5 minutes at 750 rpm.
  • RPMI media no additives
  • HAT medium RPMI/HEPES medium containing 10% Fetal Bovine Serum (JRH), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mg/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 50 mM 2-mercaptoethanol, 1% 653-conditioned RPMI media, 100 pM hypoxanthine, 0.4 pM aminopterin, and 16 pM thymidine) and then plated at 200 pL/well in five 96-well flat bottom tissue culture plates. The plates were then placed in a humidified 37°C incubator containing 5% CO2 and 95% air for 7-10 days.
  • Solid phase EIAs were used to screen mouse sera for human IgG antibodies specific for human TNFa. Briefly, plates were coated with TNFa at 1 mg/mL in PBS overnight. After washing in 0.15 M saline containing 0.02% (v/v) Tween 20, the wells were blocked with 1% (w/v) BSA in PBS, 200 pL/well for 1 hour at RT. Plates were either used immediately or frozen at -20 °C for future use. Mouse sera were incubated in two-fold serial dilutions on the human TNFa-coated plates at 50 pL/well at RT for 1 hour.
  • the plates were washed and then probed with 50 pL/well HRP-labeled goat anti -human IgG, Fc specific (Accurate) diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT.
  • the plates were again washed and 100 mL/well of the citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01% H2O2 and 1 mg/mF OPD) was added for 15 minutes at RT. Stop solution (4N sulfuric acid) was then added at 25 pF/well and the OD's were read at 490 nm using an automated plate spectrophotometer.
  • the plates were washed and probed with either HRP -conjugated goat anti-human kappa (Southern Biotech) antibody diluted 1: 10,000 in 1% BSA-HBSS or HRP -conjugated goat anti-human IgG Fc specific antibody diluted to 1:30,000 in 1% BSA-HBSS for one hour at 37°C.
  • the plates were then incubated with substrate solution as described above. Hybridoma clones that did not give a positive signal in both the anti-human kappa and anti-human IgG Fc EIA formats were discarded.
  • Isotyping Isotype determination of the antibodies was accomplished using an EIA in a format similar to that used to screen the mouse immune sera for specific titers.
  • EIA plates were coated with goat anti -human IgG (H+L) at 10 :g/mL in sodium carbonate buffer overnight at 4EC and blocked as described above. Neat supernatants from 24 well cultures were incubated on the plate for one hour at RT. The plate was washed and probed with HRP-labeled goat anti -human IgGi, IgG2, IgG3 or IgG4 (Binding Site) diluted at 1:4000 in 1% BSA-PBS for one hour at RT. The plate was again washed and incubated with substrate solution as described above.
  • GenTNV Totally Human Anti-Human TNFa Monoclonal Antibodies.
  • GenTNV One fusion, named GenTNV, was performed from a GenPharm mouse immunized with recombinant human TNFa protein. From this fusion, 196 growth-positive hybrids were screened. Eight hybridoma cell lines were identified that secreted totally human IgG antibodies reactive with human TNFa.
  • Parental cells collected from wells of a 24-well culture dish for each of the eight cell lines were handed over to Molecular Biology group on 2-18-99 for transfection and further characterization.
  • GenTNV fusion was performed utilizing splenocytes from a hybrid mouse containing human variable and constant region antibody transgenes that was immunized with recombinant human TNFa prepared at Centocor. Eight totally human, TNFa-reactive IgG monoclonal antibodies of the IgGlK isotype were generated. Parental cell lines were transferred to Molecular Biology group for further characterization and development. One of these new human antibodies may prove useful in anti-inflammatory with the potential benefit of decreased immunogenicity and allergic-type complications as compared with Remicade.
  • Example 4 Cloning and Preparation of Cell lines Expressing Human anti-TNFa antibody.
  • a panel of eight human monoclonal antibodies (mAbs) with a TNV designation were found to bind immobilized human TNFa with apparently high avidity. Seven of the eight mAbs were shown to efficiently block huTNFa binding to a recombinant TNF receptor. Sequence analysis of the DNA encoding the seven mAbs confirmed that all the mAbs had human V regions. The DNA sequences also revealed that three pairs of the mAbs were identical to each other, such that the original panel of eight mAbs contained only four distinct mAbs, represented by TNV14, TNV15, TNV148, and TNV196. Based on analyses of the deduced amino acid sequences of the mAbs and results of in vitro TNFa neutralization data, mAb TNV148 and TNV14 were selected for further study.
  • TNV148B The serine modified mAb was designated TNV148B.
  • PCR-amplified DNA encoding the heavy and light chain variable regions of TNV148B and TNV14 was cloned into newly prepared expression vectors that were based on the recently cloned heavy and light chain genes of another human mAb (12B75), disclosed in US patent application No. 60/236,827, filed October 7, 2000, entitled IL-12 Antibodies, Compositions, Methods and Uses, published as WO 02/12500which is entirely incorporated herein by reference.
  • P3X63Ag8.653 (653) cells or Sp2/0-Agl4 (Sp2/0) mouse myeloma cells were transfected with the respective heavy and light chain expression plasmids and screened through two rounds of subcloning for cell lines producing high levels of recombinant TNV148B and TNV14 (rTNV148B and rTNV14) mAbs.
  • a panel of eight mAbs derived from human TNFa-immunized GenPharm/Medarex mice were previously shown to bind human TNFa and to have a totally human IgGl, kappa isotype.
  • a simple binding assay was used to determine whether the exemplary mAbs of the invention were likely to have TNFa-neutralizing activity by evaluating their ability to block TNFa from binding to recombinant TNF receptor. Based on those results, DNA sequence results, and in vitro characterizations of several of the mAbs, TNV148 was selected as the mAh to be further characterized.
  • DNA sequences encoding the TNV148 mAh were cloned, modified to fit into gene expression vectors that encode suitable constant regions, introduced into the well- characterized 653 and Sp2/0 mouse myeloma cells, and resulting transfected cell lines screened until subclones were identified that produced 40-fold more mAh than the original hybridoma cell line.
  • TRIZOL reagent was purchased from Gibco BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse Transcriptase was obtained from Life Sciences, Inc. Taq DNA Polymerase was obtained from either Perkin Elmer Cetus or Gibco BRL. Restriction enzymes were purchased from New England Biolabs. QIAquick PCR Purification Kit was from Qiagen. A QuikChange Site-Directed Mutagenesis Kit was purchased from Stratagene. Wizard plasmid miniprep kits and RNasin were from Promega. Optiplates were obtained from Packard. 125 Iodine was purchased from Amersham. Custom oligonucleotides were purchased from Keystone/Biosource International. The names, identification numbers, and sequences of the oligonucleotides used in this work are shown in Table 2.
  • oligonucleotide 5'14s and HuH-J6 The amino acids encoded by oligonucleotide 5'14s and HuH-J6 are shown above the sequence.
  • the 'M' amino acid residue represents the translation start codon.
  • the underlined sequences in oligonucleotides 5'14s and HuH-J6 mark the BsiWI and BstBI restriction sites, respectively.
  • the slash in HuH-J6 corresponds to the exon/intron boundary. Note that oligonucleotides whose sequence corresponds to the minus strand are written in a 3 '-5' orientation.
  • a single frozen vial of 653 mouse myeloma cells was obtained.
  • the vial was thawed that day and expanded in T flasks in IMDM, 5% FBS, 2 mM glutamine (media). These cells were maintained in continuous culture until they were transfected 2 to 3 weeks later with the anti-TNF DNA described here. Some of the cultures were harvested 5 days after the thaw date, pelleted by centrifugation, and resuspended in 95% FBS, 5% DMSO, aliquoted into 30 vials, frozen, and stored for future use.
  • a single frozen vial of Sp2/0 mouse myeloma cells was obtained. The vial was thawed, a new freeze-down prepared as described above, and the frozen vials stored in CBC freezer boxes AA and AB. These cells were thawed and used for all Sp2/0 transfections described here.
  • Hybridoma cell supernatants containing the TNV mAbs were used to assay for the ability of the mAbs to block binding of 125 I-labeled TNFa to the recombinant TNF receptor fusion protein, p55-sf2 (Scallon et al. (1995) Cytokine 7:759-770). 50 :1 of p55-sf2 at 0.5 :g/ml in PBS was added to Optiplates to coat the wells during a one-hour incubation at 37°C.
  • TNV cell supernatants Serial dilutions of the eight TNV cell supernatants were prepared in 96-well round-bottom plates using PBS/ 0.1% BSA as diluent.
  • Cell supernatant containing anti-IF-18 mAb was included as a negative control and the same anti-IF-18 supernatant spiked with cA2 anti-TNF chimeric antibody, Remicade, US patent No. 5,770,198, entirely incorporated herein by reference) was included as a positive control.
  • 125 I-labeled TNFa (58 :Ci/:g, D. Shealy) was added to 100 :1 of cell supernatants to have a final TNFa concentration of 5 ng/ml. The mixture was preincubated for one hour at RT.
  • the coated Optiplates were washed to remove unbound p55-sf2 and 50 :1 of the 125 I- TNFa/cell supernatant mixture was transferred to the Optiplates. After 2 hrs. at RT, Optiplates were washed three times with PBS-Tween. 100 :1 of Microscint-20 was added and the cpm bound determined using the TopCount gamma counter.
  • Hybridoma cells were washed once in PBS before addition of TRIZOL reagent for RNA preparation. Between 7 X 10 6 and 1.7 X 10 7 cells were resuspended in 1 ml TRIZOL. Tubes were shaken vigorously after addition of 200 ml of chloroform. Samples were centrifuged at 4°C for 10 minutes. The aqueous phase was transferred to a fresh microfuge tube and an equal volume of isopropanol was added. Tubes were shaken vigorously and allowed to incubate at room temperature for 10 minutes. Samples were then centrifuged at 4°C for 10 minutes.
  • RNA pellets were washed once with 1 ml of 70% ethanol and dried briefly in a vacuum dryer.
  • the RNA pellets were resuspended with 40 ml of DEPC-treated water.
  • the quality of the RNA preparations was determined by fractionating 0.5 ml in a 1% agarose gel. The RNA was stored in a -80°C freezer until used.
  • RNA and 1 pig of either oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light chain) (see Table 1) in a volume of 11.5 ml.
  • the mixture was incubated at 70°C for 10 minutes in a water bath and then chilled on ice for 10 minutes.
  • a separate mixture was prepared that was made up of 2.5 ml of 10X reverse transcriptase buffer,
  • the unpurified heavy and light chain cDNAs were used as templates to PCR- amplify the variable region coding sequences.
  • Five oligonucleotide pairs (366/354, 367/354, 368/354, 369/354, and 370/354, Table 1) were simultaneously tested for their ability to prime amplification of the heavy chain DNA.
  • Two oligonucleotide pairs (362/208 and 363/208) were simultaneously tested for their ability to prime amplification of the light chain DNA.
  • PCR reactions were carried out using 2 units of PLATINUM TM high fidelity (HIFI) Taq DNA polymerase in a total volume of 50 ml.
  • PLATINUM TM high fidelity (HIFI) Taq DNA polymerase in a total volume of 50 ml.
  • Each reaction included 2 ml of a cDNA reaction, 10 pmoles of each oligonucleotide, 0.2 mM dNTPs, 5 ml of 10 X HIFI Buffer, and 2 mM magnesium sulfate.
  • the thermal cycler program was 95 °C for 5 minutes followed by 30 cycles of (94°C for 30 seconds, 62°C for 30 seconds, 68°C for 1.5 minutes). There was then a final incubation at 68°C for 10 minutes.
  • PCR products were purified using the QIAquickTM PCR Purification Kit according to the manufacturer’s protocol.
  • the DNA was eluted from the spin column using 50 ml of sterile water and then dried down to a volume of 10 ml using a vacuum dryer.
  • DNA sequencing reactions were then set up with 1 ml of purified PCR product, 10 mM oligonucleotide primer, 4 ml BigDye TerminatorTM ready reaction mix, and 14 ml sterile water for a total volume of 20 ml.
  • Heavy chain PCR products made with oligonucleotide pair 367/354 were sequenced with oligonucleotide primers 159 and 360.
  • the two oligonucleotides were first fractionated through a 15% polyacrylamide gel and the major bands purified. Mutagenesis reactions were prepared using either 10 ng or 50 ng of TNV148 heavy chain plasmid template (pl753), 5 ml of 10X reaction buffer, 1 ml of dNTP mix, 125 ng of primer 399, 125 ng of primer 400, and 1 ml of Pfii DNA Polymerase. Sterile water was added to bring the total volume to 50 ml.
  • reaction mix was then incubated in a thermal cycler programmed to incubate at 95 °C for 30 seconds, and then cycle 14 times with sequential incubations of 95°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, and 68°C for 7 minutes, followed by 30°C for 2 minutes (1 cycle).
  • thermal cycler programmed to incubate at 95 °C for 30 seconds, and then cycle 14 times with sequential incubations of 95°C for 30 seconds, 55°C for 1 minute, 64°C for 1 minute, and 68°C for 7 minutes, followed by 30°C for 2 minutes (1 cycle).
  • These reactions were designed to incorporate the mutagenic oligonucleotides into otherwise identical, newly synthesized plasmids.
  • samples were incubated at 37°C for 1 hour after addition of 1 ml of Dpnl endonuclease, which cleaves only the original methylated plasmid.
  • Plasmid minipreps were prepared using the WizardTM kits as described by the manufacturer. After elution of sample from the WizardTM column, plasmid DNA was precipitated with ethanol to further purify the plasmid DNA and then resuspended in 20 ml of sterile water. DNA sequence analysis was then performed to identify plasmid clones that had the desired base change and to confirm that no other base changes were inadvertently introduced into the TNV148 coding sequence.
  • One ml of plasmid was subjected to a cycle sequencing reaction prepared with 3 ml of BigDye mix, 1 ml of pUC19 Forward primer, and 10 ml of sterile water using the same parameters described in Section 4.3.
  • pl747 To modify the 12B75 heavy chain gene in plasmid pl560, a 6.85 kb BamHI/Hindlll fragment containing the promoter and variable region was transferred from pl560 to pUC19 to make pl743.
  • the smaller size of this plasmid compared to pl560 enabled use of QuikChangeTM mutagenesis (using oligonucleotides BsiWI-1 and BsiWI-2) to introduce a unique BsiWI cloning site just upstream of the translation initiation site, following the manufacturer's protocol.
  • the resulting plasmid was termed pl747.
  • a 5' oligonucleotide primer was designed with Sail and BstBI sites. This primer was used with the pUC reverse primer to amplify a 2.75 kb fragment from pl747. This fragment was then cloned back into the naturally-occurring Sail site in the 12B75 variable region and a Hindlll site, thereby introducing the unique BstB 1 site.
  • the resulting intermediate vector, designated pi 750 could accept variable region fragments with BsiWI and BstBI ends.
  • the BamHI-Hindlll insert in pl750 was transferred to pBR322 in order to have an EcoRI site downstream of the Hindlll site.
  • the resulting plasmid, pl768 was then digested with Hindlll and EcoRI and ligated to a 5.7 kb Hindlll-EcoRI fragment from pi 744, a subclone derived by cloning the large BamHI-BamHI fragment from pl560 into pBC.
  • the resulting plasmid, pl784 was then used as vector for the TNV Ab cDNA fragments with BsiWI and BstBI ends. Additional work was done to prepare expression vectors, pl788 and pl798, which include the IgGl constant region from the 12B75 gene and differ from each other by how much of the 12B75 heavy chain J-C intron they contain.
  • Any variable region fragment cloned into pi 746 would preferably be joined with the 3' half of the light chain gene.
  • oligonucleotides BAHN-1 and BAHN-2 were annealed to each other to form a double-stranded linker containing the restriction sites BsiWI, Aflll, Hindll, and Notl and which contained ends that could be ligated into Kpnl and Sacl sites. This linker was cloned between the Kpnl and Sacl sites of pBC to give plasmid pl757.
  • This new plasmid contained unique sites for BsiWI and Aflll into which the BsiWI/Aflll fragment containing the promoter and variable regions could be transferred uniting the two halves of the gene.
  • the BsiWI/BstBI inserts for TNV14, TNV148, and TNV148B heavy chains were transferred from the L28 vector to the newly prepared intermediate vector, pl750.
  • the assigned identification numbers for these intermediate plasmids are shown in Table 2. This cloning step and subsequent steps were not done for TNV15 and TNV196.
  • the variable regions were then transferred into two different human IgGl expression vectors. Restriction enzymes EcoRI and Hindlll were used to transfer the variable regions into Centocor's previously-used IgGl vector, pl04.
  • the resulting expression plasmids which encode an IgGl of the Gm(f+) allotype, were designated pi 781 (TNV14), pl782 (TNV148), and pl783 (TNV148B) (see Table 2).
  • the variable regions were also cloned upstream of the IgGl constant region derived from the 12B75 (GenPharm) gene.
  • Those expression plasmids, which encode an IgGl of the Glm(z) allotype are also listed in Table 3.
  • the L28 vector or pBC vector represents the initial Ab cDNA clone.
  • the inserts in those plasmids were transferred to an incomplete 12B75-based vector to make the intermediate plasmids.
  • transfections were distinguished by whether (1) the host cells were Sp2/0 or 653; (2) the heavy chain constant region was encoded by Centocor's previous IgGl vector or the 12B75 heavy chain constant region; (3) the mAb was TNV148B, TNV148, TNV14, or anew HC/LC combination; (4) whether the DNA was linearized plasmid or purified Ab gene insert; and (5) the presence or absence of the complete J-C intron sequence in the heavy chain gene. In addition, several of the transfections were repeated to increase the likelihood that a large number of clones could be screened.
  • Sp2/0 cells and 653 cells were each transfected with a mixture of heavy and light chain DNA (8-12 :g each) by electroporation under standard conditions as previously described (Knight DM et al. (1993 ) Molecular Immunology 30: 1443-1453).
  • the appropriate expression plasmids were linearized by digestion with a restriction enzyme prior to transfection.
  • Sail and Notl restriction enzymes were used to linearize TNV148B heavy chain plasmid pl783 and light chain plasmid pl776, respectively.
  • DNA inserts that contained only the mAb gene were separated from the plasmid vector by digesting heavy chain plasmids with BamHI and light chain plasmids with BsiWI and Noth The mAb gene inserts were then purified by agarose gel electrophoresis and Qiex purification resins. Cells transfected with purified gene inserts were simultaneously transfected with 3-5 :g of Pstl -linearized pSV2gpt plasmid (pi 3) as a source of selectable marker.
  • the highest-producing parental clones were subcloned to identify higher- producing subclones and to prepare a more homogenous cell line.
  • 96-well tissue culture plates were seeded with one cell per well or four cells per well in of IMDM, 5% FBS, 2mM glutamine, 1 X MHX and incubated at 37°C in a 5% CO 2 incubator for 12 to 20 days until colonies were apparent.
  • Cell supernatants were collected from wells that contained one colony per well and analyzed by ELISA as described above.
  • a simple binding assay was done to determine whether the eight TNV mAbs contained in hybridoma cell supernatant were capable of blocking TNFa binding to receptor.
  • concentrations of the TNV mAbs in their respective cell supernatants were first determined by standard ELISA analysis for human IgG.
  • a recombinant p55 TNF receptor/IgG fusion protein, p55-sf2 was then coated on EIA plates and 125 I- labeled TNFa allowed to bind to the p55 receptor in the presence of varying amounts of TNV mAbs. As shown in FIG. 1, all but one (TNV122) of the eight TNV mAbs efficiently blocked TNFa binding to p55 receptor.
  • TNV mAbs appeared to be more effective at inhibiting TNFa binding than cA2 positive control mAb that had been spiked into negative control hybridoma supernatant. These results were interpreted as indicating that it was highly likely that the TNV mAbs would block TNFa bioactivity in cell-based assays and in vivo and therefore additional analyses were warranted. DNA Sequence Analysis.
  • RNA was isolated from the seven hybridoma cell lines that produce these mAbs. Each RNA sample was then used to prepare human antibody heavy or light chain cDNA that included the complete signal sequence, the complete variable region sequence, and part of the constant region sequence for each mAb. These cDNA products were then amplified in PCR reactions and the PCR-amplified DNA was directly sequenced without first cloning the fragments.
  • the heavy chain cDNAs sequenced were >90% identical to one of the five human germline genes present in the mice, DP-46 (FIG. 2). Similarly, the light chain cDNAs sequenced were either 100% or 98% identical to one of the human germline genes present in the mice (FIG. 3). These sequence results confirmed that the RNA molecules that were transcribed into cDNA and sequenced encoded human antibody heavy chains and human antibody light chains.
  • variable regions were PCR-amplified using oligonucleotides that map to the 5' end of the signal sequence coding sequence, the first few amino acids of the signal sequence may not be the actual sequence of the original TNV translation products, but they do represent the actual sequences of the recombinant TNV mAbs.
  • the light chain variable region coding sequences in TNV14 and TNV 15 are identical to each other and to a representative germline sequence of the Vg/38K family of human kappa chains.
  • the TNV148 and TNV196 light chain coding sequences are identical to each other but differ from the germline sequence at two nucleotide positions (FIG. 3).
  • the deduced amino acid sequences of the four mAbs revealed the relatedness of the actual mAbs.
  • the four mAbs contain four distinct heavy chains (FIG. 4) but only two distinct light chains (FIG. 5). Differences between the TNV mAb sequences and the germline sequences were mostly confined to CDR domains but three of the mAb heavy chains also differed from the germline sequence in the framework regions (FIG. 4).
  • TNV14 was identical, TNV 15 differed by one amino acid, TNV 148 differed by two amino acids, and TNV 196 differed by three amino acids.
  • Cloning of cDNAs Site-specific Mutagenesis, and Assembly of Final Expression Plasmids. Cloning of cDNAs. Based on the DNA sequence of the PCR- amplified variable regions, new oligonucleotides were ordered to perform another round of PCR amplification for the purpose of adapting the coding sequence to be cloned into expression vectors. In the case of the heavy chains, the products of this second round of PCR were digested with restriction enzymes BsiWI and BstBI and cloned into plasmid vector L28 (plasmid identification numbers shown in Table 2).
  • the second-round PCR products were digested with Sail and AflII and cloned into plasmid vector pBC. Individual clones were then sequenced to confirm that their sequences were identical to the previous sequence obtained from direct sequencing of PCR products, which reveals the most abundant nucleotide at each position in a potentially heterogeneous population of molecules.
  • TNV148 Site-specific Mutagenesis to Change TNV148.
  • TNV148 and TNV196 were being consistently observed to be four-fold more potent than the next best mAh (TNV14) at neutralizing TNFa bioactivity.
  • TNV148 and TNV196 heavy chain framework sequences differed from the germline framework sequences.
  • a comparison of the TNV148 heavy chain sequence to other human antibodies indicated that numerous other human mAbs contained an lie residue at position 28 in framework 1 (counting mature sequence only) whereas the Pro residue at position 75 in framework 3 was an unusual amino acid at that position.
  • TNV196 heavy chain A similar comparison of the TNV196 heavy chain suggested that the three amino acids by which it differs from the germline sequence in framework 3 may be rare in human mAbs. There was a possibility that these differences may render TNV148 and TNV196 immunogenic if administered to humans. Because TNV148 had only one amino acid residue of concern and this residue was believed to be unimportant for TNFa binding, a site-specific mutagenesis technique was used to change a single nucleotide in the TNV148 heavy chain coding sequence (in plasmid pl753) so that a germline Ser residue would be encoded in place of the Pro residue at position 75. The resulting plasmid was termed pl760 (see Table 2). The resulting gene and mAh were termed TNV148B to distinguish it from the original TNV148 gene and mAh (see FIG.
  • New antibody expression vectors were prepared that were based on the 12B75 heavy chain and light chain genes previously cloned as genomic fragments. Although different TNV expression plasmids were prepared (see Table 2), in each case the 5' flanking sequences, promoter, and intron enhancer derived from the respective 12B75 genes. For the light chain expression plasmids, the complete J-C intron, constant region coding sequence and 3' flanking sequence were also derived from the 12B75 light chain gene.
  • the human IgGl constant region coding sequences derived from Centocor's previously-used expression vector (pi 04).
  • the final production cell lines reported here express a different allotype (Gm(f+)) of the TNV mAbs than the original, hybridoma-derived TNV mAbs (Glm(z)). This is because the 12B75 heavy chain gene derived from the GenPharm mice encodes an Arg residue at the C-terminal end of the CHI domain whereas Centocor's IgGl expression vector pl04 encodes a Lys residue at that position.
  • heavy chain expression plasmids e.g. pl786 and pl788
  • pl786 and pl788 were prepared in which the J-C intron, complete constant region coding sequence and 3' flanking sequence were derived from the 12B75 heavy chain gene, but cell lines transfected with those genes were not selected as the production cell lines.
  • Vectors were carefully designed to permit one-step cloning of future PCR-amplified V regions that would result in final expression plasmids.
  • PCR-amplified variable region cDNAs were transferred from L28 or pBC vectors to intermediate-stage, 12B75-based vectors that provided the promoter region and part of the J-C intron (see Table 2 for plasmid identification numbers). Restriction fragments that contained the 5' half of the antibody genes were then transferred from these intermediate-stage vectors to the final expression vectors that provided the 3' half of the respective genes to form the final expression plasmids (see Table 2 for plasmid identification numbers).
  • Expression plasmids were either linearized by restriction digest or the antibody gene inserts in each plasmid were purified away from the plasmid backbones.
  • Sp2/0 and 653 mouse myeloma cells were transfected with the heavy and light chain DNA by electroporation. Fifteen different transfections were done, most of which were unique as defined by the Ab, specific characteristics of the Ab genes, whether the genes were on linearized whole plasmids or purified gene inserts, and the host cell line (summarized in Table 4).
  • Cell supernatants from clones resistant to mycophenolic acid were assayed for the presence of human IgG by ELISA and quantitated using purified rTNV148B as a reference standard curve.
  • the identification numbers of the heavy and light chain plasmids that encode each mAh are shown.
  • plasmid p 13 (pSV2gpt) was included as a source of the gpt selectable marker.
  • the heavy chain constant regions were encoded either by the same human IgGl expression vector used to encode Remicade ('old') or by the constant regions contained within the 12B75 (GenPharm/Medarex) heavy chain gene ('new').
  • H1/L2 refers to a "novel" mAh made up of the TNV14 heavy chain and the TNV148 light chain. Plasmids pl783 and p 1801 differ only by how much of the J-C intron their heavy chain genes contain.
  • the transfection numbers which define the first number of the generic names for cell clones, are shown on the right.
  • the rTNV14-producing cell line C476A derived from transfection number 3.
  • the first digit of the original clone names indicates which transfection the cell line derived from. All of the C-coded cell lines reported here were derived from transfections with heavy and light chain whole plasmids that had been linearized with restriction enzymes. Characterization of Subcloned Cell Lines
  • TNV148B was selected as preferred based on several criteria that included protein sequence and TNF neutralization potency, as well as TNV14.
  • Cell lines were prepared that produce greaterthan 100 :g/ml of rTNV148B and 19 :g/ml rTNV14.
  • Example 5 Arthritic Mice Study using Anti-TNF Antibodies and Controls Using Single Bolus Injection
  • Tgl97 study mice were assigned, based on gender and body weight, to one of 9 treatment groups and treated with a single intraperitoneal bolus dose of Dulbecco’s PBS (D-PBS) or an anti-TNF antibody of the present invention (TNV14, TNV148 or TNV196) at either 1 mg/kg or 10 mg/kg.
  • D-PBS Dulbecco’s PBS
  • TNV14, TNV148 or TNV196 an anti-TNF antibody of the present invention
  • FIGS. 11A-C represent the progression of disease severity based on the arthritic index.
  • the 10 mg/kg cA2 -treated group’s arthritic index was lower than the D-PBS control group starting at week 3 and continuing throughout the remainder of the study (week 7).
  • the animals treated with 1 mg/kg TNV14 and the animals treated with 1 mg/kg cA2 failed to show significant reduction in AI after week 3 when compared to the D-PBS-treated Group.
  • the 1 mg/kg TNV148 showed a significantly lower AI than 1 mg/kg cA2 at 3, 4 and 7 weeks.
  • the 1 mg/kg TNV148 was also significantly lower than the 1 mg/kg TNV14-treated Group at 3 and 4 weeks.
  • TNV196 showed significant reduction in AI up to week 6 of the study (when compared to the D-PBS-treated Group), TNV148 was the only 1 mg/kg treatment that remained significant at the conclusion of the study.
  • mice were assigned, based on body weight, to one of 8 treatment groups and treated with a intraperitoneal bolus dose of control article (D-PBS) or antibody (TNV14, TNV148) at 3 mg/kg (week 0). Injections were repeated in all animals at weeks 1, 2, 3, and 4. Groups 1-6 were evaluated for test article efficacy. Serum samples, obtained from animals in Groups 7 and 8 were evaluated for immune response induction and pharmacokinetic clearance of TNV14 or TNV148 at weeks 2, 3 and 4.
  • D-PBS control article
  • TNV14, TNV148 antibody
  • FIGS. 13A-C represent the progression of disease severity based on the arthritic index.
  • the 10 mg/kg cA2 -treated group’s arthritic index was significantly lower than the D-PBS control group starting at week 2 and continuing throughout the remainder of the study (week 5).
  • the animals treated with 1 mg/kg or 3 mg/kg of cA2 and the animals treated with 3 mg/kg TNV14 failed to achieve any significant reduction in AI at any time throughout the study when compared to the d-PBS control group.
  • the animals treated with 3 mg/kg TNV148 showed a significant reduction when compared to the d-PBS-treated group starting at week 3 and continuing through week 5.
  • the 10 mg/kg cA2 -treated animals showed a significant reduction in AI when compared to both the lower doses (1 mg/kg and 3 mg/kg) of cA2 at weeks 4 and 5 of the study and was also significantly lower than the TNV14-treated animals at weeks 3-5. Although there appeared to be no significant differences between any of the 3mg/kg treatment groups, the AI for the animals treated with 3 mg/kg TNV14 were significantly higher at some time points than the 10 mg/kg whereas the animals treated with TNV 148 were not significantly different from the animals treated with 10 mg/kg of cA2.
  • Tgl97 study mice were assigned, based on gender and body weight, to one of 6 treatment groups and treated with a single intraperitoneal bolus dose of antibody (cA2, or TNV148) at either 3 mg/kg or 5 mg/kg.
  • This study utilized the D-PBS and 10 mg/kg cA2 control Groups.
  • FIG. 15 represents the progression of disease severity based on the arthritic index. All treatment groups showed some protection at the earlier time points, with the 5 mg/kg cA2 and the 5 mg/kg TNV 148 showing significant reductions in AI at weeks 1-3 and all treatment groups showing a significant reduction at week 2. Later in the study the animals treated with 5 mg/kg cA2 showed some protection, with significant reductions at weeks 4, 6 and 7. The low dose (3 mg/kg) of both the cA2 and the TNV148 showed significant reductions at 6 and all treatment groups showed significant reductions at week 7. None of the treatment groups were able to maintain a significant reduction at the conclusion of the study (week 8). There were no significant differences between any of the treatment groups (excluding the saline control group) at any time point.
  • Example 8 Arthritic Mice Study using Anti-TNF Antibodies and Controls as Single Intraperitoneal Bolus Dose Between Anti-TNF Antibody and Modified Anti-TNF Antibody
  • TNV148 derived from hybridoma cells
  • rTNV148B derived from transfected cells
  • FIG. 17 represents the progression of disease severity based on the arthritic index.
  • the 10 mg/kg cA2 -treated group’s arthritic index was lower than the D-PBS control group starting at week 4 and continuing throughout the remainder of the study (week 8).
  • Both of the TNV148-treated Groups and the 1 mg/kg cA2 -treated Group showed a significant reduction in AI at week 4.
  • a previous study (P-099-017) showed that TNV148 was slightly more effective at reducing the Arthritic Index following a single 1 mg/kg intraperitoneal bolus, this study showed that the AI from both versions of the TNV antibody-treated groups was slightly higher.
  • Example 9 Anti-TNF Antibodies for the Treatment or Prevention of Type 1 Diabetes (T1D)
  • SIMPONI ® prolimumab administered subcutaneously (SC) in the treatment of Type 1 diabetes (T1D) to determine whether benefit with SIMPONI ® can be established in newly diagnosed T1D patients in a Phase 2a efficacy and safety study was requested.
  • This study in a newly diagnosed population would be an initial step in the development program to understand whether SIMPONI ® would have benefit in pre-TID patients to stabilize endogenous insulin production to prevent or delay disease progression.
  • SIMPONI ® is a human monoclonal antibody (mAb) with an immunoglobulin G (IgG) 1 heavy chain isotype (Glm [z] allotype) and a kappa light chain isotype.
  • Golimumab has a heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37.
  • Golimumab binds with high affinity to both soluble and transmembrane forms of tumor necrosis factor alpha (TNFa) and inhibits TNFa bioactivity.
  • Golimumab is classified according to the Anatomical Therapeutic Chemical (ATC) Classification System as a TNFa inhibitor (ATC code: F04AB06).
  • ATC Anatomical Therapeutic Chemical
  • Golimumab is approved under the trade name of SIMPONI ® in the United States (US) and atotal of 89 countries worldwide as of 6 October 2015 for the treatment of rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) as a 50 mg SC injection administered once a month as well as in 67 countries for the treatment of ulcerative colitis (UC) as a 200 mg SC injection at Week 0, followed by 100 mg at Week 2 induction regimen, and maintenance therapy with 100 mg every 4 weeks thereafter.
  • SIMPONI ® is approved for the following indications:
  • the Sponsor intends to use 2 dosage forms in the initial Phase 2a study to support the proposed doses across the target population. These 2 dosage forms are described below:
  • Each 50 mg single dose prefdled glass syringe (27 gauge 1 ⁇ 2 inch needle) contains 50 mg of SIMPONI ® per 0.5 mL of solution.
  • VARIOJECT® injector
  • BSA body surface area
  • T1D is one of the three most prevalent chronic diseases of childhood and approximately 3 million people have this disease (Daneman 2006, Stanescu, Lord et al. 2012). The annual incidence is highest in children and adolescents, at ⁇ 20 cases/100, 000/year in those younger than 20 years old, accounting for nearly 15,000 new cases yearly. (Maahs, West et al. 2010, Stanescu, Lord et al. 2012) In the U.S. and worldwide, the incidence of T1D is on the rise, with increases of -3-5% per year in most areas (Daneman 2006, Stanescu, Lord et al. 2012).
  • T1D In the US, between 2001 and 2009, the prevalence of T1D in children and adolescents rose by 23%.(JDRF 2013) The diagnosis of T1D increases during childhood and peaks in adolescence: in those 0-4, 5-9, 10-14, and 15-19 years old annual rates are 14, 22, 26, and 13 cases/100,000, respectively (Stanescu, Lord et al. 2012). Comparatively, T1D rates are much lower in adults, with the incidence only up to -8/100,000 (Molbal., Christau et al. 1994).
  • DKA diabetic ketoacidosis
  • T1D results from an autoimmune attack on the pancreatic beta cells
  • a proinflammatory combination of innate and adaptive and cellular and humoral responses is responsible for T1D (Bluestone, Herold et al. 2010).
  • T1D is hypothesized to occur in predisposed individuals who encounter a diabetogenic environmental trigger.
  • a number of HLA- and other immune system-associated genes are linked to T1D susceptibility (van Belle, Coppieters et al. 2011).
  • T1D Those with T1D appear to have abnormalities in both central and peripheral tolerance mechanisms that involve b- ce 11 -reactive T cells (Eisenbarth 2004). Although both dietary and infectious factors have been implicated, none have been found to be causally associated with T ID. It is hypothesized that some environmental encounter activates an inflammatory response, that in predisposed individuals, results in the activation and recruitment of macrophages, dendritic cells, CD4 and CD8 T cells, and B cells to islets (Bluestone, Herold et al. 2010, Atkinson, Bluestone et al. 2011). Soluble factors, including proinflammatory cytokines and beta cell autoantibodies, participate in and amplify this response, and the end result is terminal destruction of beta cells.
  • DCCT Diabetes Control and Complications Trial
  • hypoglycemia varies considerably among studies, with greater incidence in both symptomatic and severe hypoglycemia observed in subjects (Cryer 1989). Furthermore, studies using continuous blood glucose measurements over prolonged periods have generally found that the frequency and duration of hypoglycemia, especially the nocturnal hypoglycemia, is even greater than what was previously thought (Guillod 2007, Wentholt 2007). Another factor that prevents patients with T1DM from achieving adequate glycemic is undesirable weight gain which often occurs as a consequence of intensive insulin treatment, with a subsequent reduction of glycosuria.
  • T2DM Type 2 Diabetes Melbtus
  • insulin remains the mainstay of therapy for patients with T1DM.
  • various types of insulin have been developed, ranging from the traditional insulins to the more modem insulin analogues, with insulin lispro being the first short acting analogue approved by the FDA in 1996, and glargine the first long -acting analogue approved in 2000. Since then, other short and long-acting analogues have been approved and their use has become increasingly common. This is in part due to the need for achieving near euglycemia in subjects with T1DM and the marked increase in risk for severe hypoglycemia seen with non-analogue insulins.
  • NPH Neutral Protamine Hagedom
  • these analogues provide greater flexibility and convenience to the patients, along with better compliance, as the short-acting analogues may be injected immediately before or even after a meal (compared with 30 minutes prior to meals for regular insulin) and the long- acting analogues once-daily (compared to twice daily for NPH).
  • DCCT 1993 the DCCT was a large study conducted in subjects with T1D to evaluate the potential benefits of intensive insulin therapy compared with standard insulin therapy on the development and progression of long-term complications.
  • DCCT 1993 subjects who retained significant residual b-cell function (as measured by C-peptide >0.2 pmol/mL) were compared with subjects who did not retain significant b-cell function (C-peptide ⁇ 0.2 pmol/mL).
  • C-peptide ⁇ 0.2 pmol/mL the DCCT 1987; DCCT 1998; Steffes 2003; Palmer 2004.
  • T1D The notion that maintaining endogenous insulin production has important short- and long-term benefits in those with T1D has been some of the most important justification for numerous interventional trials in new onset T1D in the past 1 to 2 decades. Although an ultimate goal in this field may be “full” remission of T1D (and thus insulin independence), a more realistic, and likely a more achievable goal is to prevent destruction of b-cells present at diagnosis (often considered to be -10-20% of baseline numbers). Even though individuals may still require some exogenous insulin, metabolic control would be improved, and T ID-associated complications would be lessened. Recent data in T1D have supported this concept.
  • Tumor necrosis factor alpha is a principal proinflammatory cytokine produced primarily by macrophages and T cells in response to a variety of stimuli and mediates a wide range of biological activities. It is expressed as a 26 kilodalton (kDa) type II membrane protein that, upon proteolysis, is released as a soluble 17 kDa monomer that self-associates into the biologically active trimeric form.
  • Tumor necrosis factora is part of the TNF ligand superfamily, a group of related cytokines with overlapping functions that influence cell proliferation and cell death in processes ranging from development to immune response. As indicated by its name, TNFa was initially described as an inducer of apoptosis with murine tumor cell lines, but more recent studies suggest chronic inflammation due to TNFa can lead to tumor promotion and metastasis.
  • TNFa The functional activities modulated by TNFa can include cell activation leading to proliferation, differentiation, induction of cytokine and chemokine production and induction of adhesion proteins, or initiation of programmed cell death.
  • the 2 receptors that engage TNFa are found on virtually all cell types.
  • TNF receptor 1 (TNF-R1) (or p55) contains an intracellular death domain and can signal cytotoxic events, while TNF receptor 2 (TNF-R2) (or p75) appears to be involved in TNFa signaling in lymphocytes.
  • TNFa physiological response to TNFa production
  • TNFa biological form of TNFa present (soluble or transmembrane), the functional receptor (TNF-Rl and/or TNF-R2), accessory proteins, and signaling pathways available in the target cell, the tissue in which it is produced, and the timing and duration of expression.
  • TNFa While limited, local, expression of TNFa is important in the host inflammatory and protective immune response to injury and infectious pathogens, chronic expression of TNFa in specific organs can lead to significant pathology. High levels of TNFa have been implicated in the pathophysiology of diseases such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), inflammatory bowel disease and T1D.
  • RA rheumatoid arthritis
  • PsA psoriatic arthritis
  • AS ankylosing spondylitis
  • T1D inflammatory bowel disease
  • TNFa agents including the monoclonal antibodies (mAbs) golimumab, infliximab, adalimumab, and certolizumab pegol; and the soluble TNF receptor p75 fragment crystallizable (Fc) fusion protein, etanercept, for the treatment of immune-mediated inflammatory diseases such as RA, inflammatory bowel disease, and adjacent indications.
  • mAbs monoclonal antibodies
  • Fc soluble TNF receptor p75 fragment crystallizable
  • TNFa is a proinflammatory cytokine is critical in the autoimmune process and beta cell destruction in TlD.
  • Tumor necrosis factor-a produced by activated macrophages, dendritic cells, and CD4+ T cells promotes inflammation via its participation in the acute phase response, pro-proliferative effects, and activation and recruitment of other cells in the innate and adaptive immune response.
  • Tumor necrosis factor-a, dendritic cells (DCs), monocytes, and CD4+ T cells are all found in the insulitic lesion in T1D, and all are implicated in beta cell inflammation (insulitis) and beta cell killing.
  • Tumor necrosis factor-a appears to promote diabetes autoimmunity by enhancing the recruitment of inflammatory cells to the islets, activating cells and enhancing autoantigen presentation.
  • Tumor necrosis factor-a activates vascular endothelium, upregulating MHC I and adhesion molecules.
  • DCs dendritic cells
  • Tumor necrosis factor-a has direct cytostatic effects and impairs insulin production and secretion, and it has cytocidal activity, killing beta cells directly.
  • Tumor necrosis factor-a also impairs insulin signaling and increases peripheral insulin resistance, which in experimental models can be reversed by blocking TNFa.
  • TNFa also has potent metabolic effects that may contribute to diabetes by increasing beta cell stress and death.
  • Eligibility included patients aged 3-18 years, ⁇ 6 weeks from diagnosis, who were diabetes autoantibody (i.e., GAD-65 and/or islet cell antibody) positive, had normal WBC and platelet counts, and normal liver and renal function. Patients received etanercept 0.4 mg/kg (maximum 25 mg) dosed subcutaneously twice weekly for 24 weeks or placebo.
  • TNFa has a critical role in T1D development and progression.
  • TNFa appears to play a key role in disease initiation and progression.
  • the clinical utility of TNFa blockade in other autoimmune disorders is well established.
  • FDA-approved agents to block TNFa including adalimumab, golimumab, infliximab, certolizumab pegol and etanercept, and this class of biologic therapies has been the most extensively evaluated, prescribed and, have been successful in the treatment of a spectrum of autoimmune diseases in adults and children such as rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis and Crohn’s disease.
  • TNFa inhibitors There are a number of off-label and experimental uses for TNFa inhibitors. This includes allogeneic islet transplant regimens, where adding TNFa inhibitors have shown to improve graft survival, perhaps reflecting both beneficial immunologic and metabolic effects of blocking TNFa on beta cell survival in this indication.
  • Golimumab, infliximab, certolizumab pegol and adalimumab are monoclonal antibodies that bind TNFa; whereas etanercept is a fusion protein consisting of the TNF receptor bound to an IgG tail and also binds lymphotoxin alpha (LTa). Lymphotoxin alpha appears to have a role in rodent autoimmune diabetes, but has not been shown to be directly involved in human T1D.
  • TNFa in the immune and metabolic pathogenesis of T1D
  • blocking TNFa has the ability to interfere with diabetes autoimmunity and preserve beta cells.
  • TNFa-blockers There is almost 2 decades of successful clinical experience of TNFa-blockers with in a variety of human autoimmune diseases, including in children as young as 2 years old.
  • T1D continues to be a significant burden on individuals, their families and society and there is a significant unmet need for a disease modifying therapy in T1D which can assist in maintaining endogenous beta cell function and lessen the short- and long-term sequelae of this disease.
  • SIMPONI ® (golimumab) administered as a SC injection is currently approved in the US for adults for the treatment of moderately to severely active rheumatoid arthritis (RA) in combination with methotrexate (MTX); active psoriatic arthritis (PsA), alone or in combination with MTX; active ankylosing spondylitis (AS); and moderately to severely active UC in patients with an inadequate response to or intolerance of prior treatments or requiring continuous steroid therapy.
  • RA rheumatoid arthritis
  • MTX methotrexate
  • PsA active psoriatic arthritis
  • AS active ankylosing spondylitis
  • the approved dosage in the adult rheumatology indications is 50 mg administered by SC injection once a month.
  • the approved dosage for adults with UC is a 200 mg SC injection at Week 0 followed by 100 mg SC at Week 2 as induction (200 mg 100 mg), followed by maintenance therapy of 100 mg SC q4w.
  • the ADR frequencies in Error! Reference source not found, are based on data from 5,717 golimumab-treated subjects in the Phase 2 and 3 clinical studies: 3,090 subjects in RA studies (C0524T02, C0524T05, C0524T06, C0524T11, C0524T28, C0524T12, and CNT0148ART3001), 394 subjects in the PsA study (C0524T08), 564 subjects in the AS studies (C0524T09, C0524T29), 1,245 subjects in UC studies (C0524T16, C0524T17, and C0524T18), 231 subjects in the severe, persistent asthma study (C0524T03), and 193 subjects with active nr-axial SpA (P07642 [MK-8259- 006]).
  • the ADRs listed in the table below are classified according to frequency and SOC.
  • the frequency categories are defined in the footnote of the table as Very common, Common, Uncommon, Rare, Very rare, and Not Known.
  • Golimumab is not currently approved for the use in pediatric patients.
  • a study of SC golimumab in children with pJIA (CNT0148JIA3001) was conducted and a clinical development program with subcutaneously administered golimumab in pediatric UC is currently ongoing (CNT0148UC01001).
  • CNT0148JIA3001 was a Phase 3, multicenter, double-blind, randomized withdrawal study with the primary objective to assess the clinical efficacy of SC administration of golimumab in pediatric subjects (ages 2 to less than 18 years) with pJIA manifested by >5 joints with active arthritis despite methotrexate (MTX) therapy for >3 months.
  • MTX methotrexate
  • the CNT0148JIA3001 study consisted of an open label phase where patients received SC golimumab 30 mg/m 2 q4w + MTX for 16 weeks, followed by a randomized withdrawal phase where patients who achieved an American College of Rheumatology (ACR) pediatric (Ped) 30 response at Week 16 received either golimumab 30 mg/m 2 + MTX or placebo + MTX q4w through Week 48. There was also a long-term extension in which the median follow-up was approximately 2 years.
  • a total of 173 subjects (75.7% female; 24.3% male) were enrolled in the study, and 154 subjects entered the randomized withdrawal phase at Week 16.
  • the mean age was 11.2 years (52.0% aged 12 to 17 years; 48.0% aged 2 to 11 years) and the mean weight was 43.1 kg.
  • CNT0148UC01001 was a Phase lb, multicenter, open-label study to assess the PK, safety, and efficacy of golimumab in children with pediatric UC.
  • This multicenter, open-label study enrolled pediatric subjects aged 2 through 17 years with moderately to severely active UC who demonstrated an inadequate response to, failed to tolerate, or had medical contraindication to conventional therapies (i.e., IV or oral corticosteroids or the immunomodulators AZA or 6-MP), and were naive to anti-TNFa agents.
  • the study was divided into 2 parts: the PK portion through Week 14, and the study extension through Week 126.
  • the 14-Week PK portion of Study 1 (CNT0148UC01001) has been completed and the study extension is ongoing.
  • a total of 35 subjects (51.4% female; 48.6% male) were enrolled in study.
  • the mean age was 13.4 years (71.4% aged 12 to 17 years; 28.6% aged 2 to 11 years) and the mean weight was 51.7 kg.
  • the results of this study provide key information on whether SIMPONI ® (golimumab) via TNFa blockade impacts T1D disease progression and support further development in new onset disease as well as in a “pre-disease” state, prior to onset of symptoms and requirement for exogenous insulin.
  • the key aspects of this study and some findings are outlined below.
  • Study subjects who weigh >45 kg who were randomized to the golimumab treatment group received an induction dose of SC golimumab 100 mg at Weeks 0 and 2 followed by a maintenance dose of SC golimumab 50 mg at Week 4 and q2w through Week 52 (Section 0).
  • Subjects randomized to the placebo treatment group received a SC placebo injection q2w through Week 52 (FIG. 18).
  • the Sponsor allowed self-administration of study agent at home.
  • HbAlc targets as defined by the American Diabetes Association. These current recommendations are intended to achieve glucose levels that appear to decrease some of the short- and long-term sequela of T1D. Specific to this study, this includes a HbAlc target of less than 8% for those aged 6 to 12 years, less than 7.5% for those between from age 13 through 18 years, and less than 7% for those 19 years old and above.
  • the subject’s glycemic control was monitored by the subject’s primary care physician.
  • HbAlc and other parameters of glycemic control were assessed at screening, and during the study.
  • T1D Males and females 6 through 21 years of age with newly diagnosed T1D were enrolled in this study. Participants met accepted criteria for enrollment in new onset T1D studies including meeting the current ADA definition of T1D, being positive for at least 1 of 5 recognized T1D autoantibodies, showing evidence of residual b-cell function (defined by a peak c-peptide of at least 0.2 pmol/mL following a mixed-meal tolerance test), and randomization in the trial within 100 days of T1D diagnosis. Exclusion criteria focused on identifying individuals who may have been at any particular risk due to immune or infectious risks if included in the trial.
  • the age range was chosen for the following reasons:
  • alefacept LFA3-Ig
  • abatacept CLA4-Ig
  • thymoglobulin appears to have a beneficial effect only in older individuals (>21 years of age;
  • Golimumab has been studied in over 200 children ages 2 years old and above with JIA and is currently under registration for this indication in Europe.
  • all of the above agents are approved for a wide variety of autoimmune conditions in adults, including rheumatoid arthritis, Crohn’s disease, ulcerative colitis and psoriasis.
  • TNFa-blocking agents including golimumab, are well established not only in adults, but specifically the pediatric population, likely much more than any other immunotherapy that has been evaluated in T1D.
  • TNFa-blockade has the ability to slow, if not reverse, the loss of b-cells in those newly diagnosed T1D, thus meeting a critical bar of a “prospect of benefit” of TNFa-blockade in this age group.
  • T1D diabetes is different in younger versus older individuals and that there can be a significantly different responses to immune therapy in these groups, where efficacy (or lack thereof) in one group may actually be misinformative to the other.
  • the Sponsor choose the upper age “cut-off’ as age 21 , as many of these studies appear to show a differentiating break in the “younger” and “older” disease at this age.
  • Age 6 has been an accepted lower age of enrollment in other new onset T1D studies (i.e., abatacept and canakinumab) and is also the age cutoff for approved UC in children and inclusive of the JIA age range for the class of approved TNFa-blockers.
  • golimumab in this trial integrates knowledge regarding disease-specific considerations in T1D, an understanding of the comparative efficacy of TNFa inhibition of etanercept in the aforementioned pilot trial in children with T1D, and the sponsor’s experience with golimumab.
  • T1D the destruction of b-cells is irreversible and appears to be rapid at the onset of clinical disease.
  • higher or more frequent induction doses followed by lower maintenance doses are often used, such as with the use of anti -TNFa agents for Crohn’s disease and ulcerative colitis.
  • T1D For T1D, there is also a need to quickly suppress disease activity to prevent further destruction of b-cell present at enrollment in study. Given that golimumab steady-state concentrations are generally established after 12 weeks, induction doses should be employed followed by a maintenance dosing regimen in order to achieve steady-state concentrations earlier to offset further b-cell loss. Data from previous adult and pediatric studies were evaluated along with population PK and mechanistic PK/target engagement (TE) modeling to determine the proposed dosing regimen for this Phase 2a new onset T1D study.
  • TE mechanistic PK/target engagement
  • the population PK model for golimumab was based on an established polyarticular JIA model in which all subjects were on concomitant methotrexate (MTX). Since MTX has been previously shown to affect golimumab exposure 5 and patients with T1D are not expected to be concomitantly treated with methotrexate, a 36% increase in golimumab clearance was accounted for in the simulations (FIG. 19).
  • MTX methotrexate
  • the 30 mg/m 2 dose is designed to be similar to the 50 mg dose in adults and the 60 mg/m 2 dose to be similar to the 100 mg dose in adults.
  • PK exposure for the proposed T1D dosing regimen is expected to be between the JIA and ped UC exposure (both simulated without MTX), though the q2w maintenance dosing interval will result in slightly higher trough concentrations.
  • golimumab 50 mg q4w was the minimum effective dosing regimen for the treatment of RA, PsA, or AS.
  • the mechanistic PK/TE model incorporated PK exposure from the above population PK model (with 36% higher clearance without concomitant MTX) paired with a target-mediated drug disposition (TMDD) model was used to assess the interaction between drug and target and to simulate the suppression of TNFa after anti- TNFa administration (FIG. 20).
  • the PK/TE model was developed based on the assumption that the etanercept dosing regimen tested in T1D results in adequate TNFa suppression given the positive results previously observed. 6
  • the TNFa kinetic parameters were obtained from preclinical studies and allometric scaling, and the same set of TNFa kinetics parameters were used to compare the effect of golimumab and etanercept.
  • TNFa suppression in the systemic circulation was also assumed to be representative of that in the pancreas.
  • the golimumab dosing regimen was designed to approximate the extent of TNFa suppression following the etanercept dosing regimen in the pilot trial in children with new onset TID, 6 (Mastrandrea, 2009), after accounting for the differences in PK and TNFa binding affinity between golimumab and etanercept.
  • the PK/TE model suggested that an induction dose regimen of 60 mg/m 2 SC (to a maximum of 100 mg) at Week 0 and Week 2 followed by a maintenance dose regimen of 30 mg/m 2 SC (to a maximum of 50 mg) q2w or 60 mg/m 2 SC (to a maximum of 100 mg) q4w allows suppression of TNFa to a level closer to approximating that of etanercept, in contrast to the 30 mg/m 2 SC q4w maintenance dose (FIG. 20).
  • the 30 mg/m 2 q2w and 60 mg/m 2 q4w maintenance dose regimens would have the same overall exposure (AUC) and similar TNFa suppression; however, the 60 mg/m 2 q4w regimen would have higher peak/trough concentration fluctuation and thus more fluctuation on suppression of TNFa. It is known that TNFa has direct cytostatic and cytocidal effects on beta cells. Thus, the TNFa elevations that would be expected to occur during the troughs with the higher, but less frequent dosing, may be damaging to residual beta cells.
  • the 30 mg/m 2 SC (to a maximum of 50 mg) q2w dose regimen was preferred as the simulations have shown the q2w maintenance dosing interval will result in slightly higher trough concentrations resulting in a smaller peak/trough fluctuation in an attempt to optimally protect b-cells from the direct effects of TNFa.
  • golimumab has been extensively characterized in subjects with RA, PsA, AS and UC.
  • 4 different dosing regimens were evaluated (50 mg q2w or q4w and 100 mg q2w or q4w) of which all doses tested were generally well tolerated and effective in maintaining clinical response through Week 52.
  • pJIA a 30 mg/m 2 q4w dosing regimen was studied up to a maximum of 50 mg (the approved adult RA dose).
  • the proposed T1D dosing regimen will achieve golimumab exposures observed between the aforementioned JIA and UC dosing in children (which the Agency has supported for study in overlapping age ranges) and thus mitigate safety concerns with this specific approach. If this trial is successful, considerations for dose ranging will be explored in subsequent clinical studies.
  • the major trial endpoints in study CNT0148DML2001 were consistent with those used and accepted by leading T1D research networks (including T1D TrialNet and the Immune Tolerance Network) and cited by major health authority guidelines (including those of the FDA and EMA). Specifically, the primary endpoint was to stimulate c-peptide response (4h AUC) following a mixed-meal tolerance test at Week 52, as an objective measure of endogenous insulin production. As this was a placebo- controlled trial, a positive study was defined as showing a statistically significant difference in the c-peptide AUC in the active versus placebo treatment groups at Week 52. Provoked c-peptide evaluations were also conducted at approximately Weeks 13,
  • the goal of this project was to determine if there was a beneficial effect on the progression of T1D, and although not expected, the Sponsor was able to use the evaluations for this to determine if there are endocrinologic adverse effects, such as increased rates of hypoglycemia, poorer glycemic control or more rapid loss of endogenous beta cell function.
  • DMC Data Monitoring Committee
  • the option for at-home administration was expected to aid in a patient’s routine (i.e., not needing to visit the site of care for every dose) and also increase study enrollment and retention. If a patient or caregiver was to perform at home administration, he/she were instructed in injection techniques, and their ability to inject subcutaneously was assessed to ensure the proper administration. In addition, it was recommended that the first self-injection or caregiver injection be performed under the supervision of a qualified healthcare professional.
  • golimumab were administered subcutaneously using the 50 mg PFS-U device, which is already approved for use in adults. For additional details, see below and FIG. 21.
  • the ULTRASAFE PASSIVE® (needle guard) is a manually-operated, single use, disposable needle guard system that is an accessory to a prefilled syringe and is intended for use as a safety mechanism to reduce the occurrence of accidental needlesticks to healthcare professionals and patients or their caregivers after administration and during disposal of a used prefdled syringe.
  • the ULTRASAFE PASSIVE® (needle guard) accepts either a 0.5 mL or a 1.0 mL PFS. There is no direct drug product contact with the device whatsoever, either during assembly or use.
  • the device clear plastic construction and the design of the viewing slot permit visualization of the syringe.
  • the passive nature of the device permits normal needle insertion and when the plunger stopper of the syringe is fully advanced and the drug dose is delivered, the spring-aided guard is released. The guard automatically advances over the syringe and needle as the user relaxes their grip until it latches in a locked position.
  • the Sponsor is developing a pediatric presentation known as the VARIOJECT® (injector) as a platform device across multiple pediatric programs to facilitate BSA-dosing, and is planning to utilize the VARIOJECT® (injector) device for this study.
  • the Sponsor has previously discussed the data required to support the registration of the VARIOJECT® (injector) device in other pediatric programs for SIMPONI ® (golimumab) which include actual use and label comprehension assessments, data from a human factor study, and additional device performance data.
  • Dosage and device selection charts (Table 8) will be developed to allow healthcare providers, caregivers, and/or pediatric subjects (as applicable) to determine the corresponding absolute milligram (mg) dose and the combination of injection devices to be used.
  • the VARIOJECT® (injector) device would be intended for delivery of a single dose of drug, based on the BSA dose regimen, ranging from doses of 10 mg to 45 mg, in 5 mg increments.
  • the VARIOJECT® (injector) device is designed to be assembled with the same 1 mL Becton Dickson HYPAK® (syringe) containing 0.5 mL of SIMPONI® (golimumab) drug product (PFS) that has been used in the already approved SIMPONI® (golimumab) SMARTJECT® (autoinjector) and PFS-U. Note that the VARIOJECT® (injector) device has no contact with the drug product, and therefore, the VARIOJECT® (injector) device has no effect on the biochemical properties or stability of the drug product.
  • VARIOJECT® (injector) device has been developed as a platform technology by Ypsomed, Holding AG, Switzerland, an experienced supplier of prefilled pens for other indications.
  • the device has been designed in accordance with the design control requirements of the Quality System regulation, 21 CFR Part 820.
  • VARIOJECT® (injector) device The overall configuration of the VARIOJECT® (injector) device and its features are depicted in FIG. 22.
  • FIG. 23 shows the device at the different stages of use: the device is primed, dose settings are selected by turning the plunger to the desired fixed dose, and the dose is administered by pushing the plunger.
  • the user first removes the cap (FIG. 24, Step B), then primes the VARIOJECT® (injector) by tapping the bubbles (visible though the viewing window as shown in FIG. 24, Step C) to the top of the syringe and pressing on the plunger to remove the air (FIG. 24, Step C).
  • the user dials the plunger to select the appropriate dose (FIG. 24, Step D).
  • the user presses the device against the skin at approximately a 90 degree angle, causing the needle guard to retract and the needle to be inserted into the selected SC injection site (FIG. 24, Step E), and then pushes the plunger to deliver the dose (FIG. 24, Step F).
  • the user removes the device, allowing the needle guard to passively extend and lock over the needle, offering protection against accidental needle sticks (FIG. 24, Step F).
  • the plunger locks in the down position, preventing reuse of the device.
  • the pen is designed to deliver between 0.10 mF to 0.45 mF in 0.05 mF increments.
  • the requirement on dosage accuracy was established based on the ISO 11608-1 2012 Needle-based injection systems for medical use, Requirements and test methods, Part 1: Needle-based injection systems, and USP 31 General Requirements/Injections.
  • the technical design requirement for delivery accuracy is that the pen must deliver the dialed dose -0.00/+0.05 mF, where 0.05 mF is the minimum increment.
  • the needle protrusion distance of a nominal 4.5 mm limits the injection depth to subcutaneous tissue.
  • the device has a number of features to help ensure that it is used properly and safely.
  • An orange priming band and white arrow indicating that the plunger should be pushed serve to remind the user that the device must be primed before use.
  • the orange priming band disappears post-priming, indicating that this step has been completed, and the dose cannot be selected until the device has been primed.
  • Graphics on the plunger align with the dose selection notch, clearly indicating the dose that is being selected, and detents provide tactile feedback to the user that the device is properly aligned.
  • the plunger locks in the down position, and the dose that was delivered locks into the dose notch, both confirming and providing a record of the dose that was delivered. Additionally, the lock safeguards against the potential for unauthorized reuse of leftover product.
  • the needle guard automatically extends and locks out. This passive needle safety feature aids in reducing the potential for accidental needle sticks and also minimizes the visual exposure of the needle to some patients and caregivers who may have a fear of needles.
  • Testing included bench tests ensuring accurate delivery of the drug product as well as other suitability and Human Factors studies.
  • the Sponsor In preparation for clinical studies, the Sponsor has completed all verification and validation testing to assess safety, usability, and performance of the VARIOJECT® (injector) device. Although the Sponsor does not anticipate any significant design changes to the device used for clinical studies, the Sponsor does intend to enhance the robustness of the VARIOJECT® (injector) design based on findings during routine development efforts.
  • the Simulated Use Safety study although the study successfully demonstrated the functionality of the sharps injury prevention feature of the device, it was noted that there were rare instances where select users, using very high forces exceeding typical delivery forces (>70 N), were capable of overcoming the maximum push-through forces during priming and dosing the device.
  • VARIOJECT® injector injections each (557 devices total among 18 participants) into a pad at a rapid rate, which is not considered representative use when injecting patients where users would take their time and excessive force would not be used. Note that this failure had not been observed previously in any of the multiple human factors studies performed with the device.
  • the Sponsor intends to pursue minor design enhancements to further strengthen the priming and dosing end stop features by increasing the overlapping area between the contacting end stop surfaces to mitigate the risk of administering the incorrect dose to the patient.
  • the current specification for the priming end stop is 46 N and the specification for the dosing end stop is 69 N.
  • the dosing end stop specification is 1.5xthe priming end stop specification due to risk of an overdose resulting from failure of the dosing end stop. Verification tests of these features show that the current design exceeds the specifications by about 50% for priming and 35% for dosing. Based on results from the Simulated Use Safety study, the Sponsor is increasing the specifications and implementing minor design changes to increase the force required to push past the priming and dosing end stops. The proposed modifications will have no impact to the user interface (all forces to operate and use steps remain unchanged) and therefore, the device used clinically will be representative of the commercial device.
  • Dose button will increase in diameter by 0.5- 1.0 mm.
  • End cap will increase in diameter by 0.5-1.0 mm.
  • the improvements will increase the robustness of the device’s performance while mitigating risks of incorrect dosing to the patient, without changing the user interface.
  • appropriate training will be provided to minimize the potential for pushing past the end stop.
  • the injection characteristics of the VARIOJECT® are similar in injection depth and duration to those performed by a healthcare provider using a manually injected subcutaneous hypodermic needle. Additionally, as described above, this manual injector has been designed with a number of features to help ensure that it is used properly and safely in pediatric patients and includes a passive needle safety feature. This important safety feature aids in reducing the potential for accidental needle sticks and also minimizes the exposure of the needle to some young patients who may have a fear of needles.
  • the needle insertion depth of this device is only 4.5 mm, designed to limit the injection to subcutaneous tissue in pediatric patients. Additionally, similar to an insulin pen, this device is suitable for at-home administration by caregivers and patients, including self-administration by appropriately trained pediatric patients capable of self-administration. An overview of the risk management activities related to in-home-use and needle length, are discussed below.
  • VARIOJECT® injector
  • the Sponsor conducted ethnographic research in the homes of children who require injections, primarily insulin and human growth hormone. Insights were gleaned from this research that led to design improvements to help ensure safe and effective use of VARIOJECT® (injector) in the home environment, and these improvements were subsequently confirmed to be effective in human factors studies. Examples include:
  • Examples include:
  • the VARIOJECT® (injector) and its associated instructions for use also have several safety features, as well as features to improve overall usability that facilitate in home-use.
  • the Sponsor will conduct a pediatric Human Factors validation study designed to evaluate in-home-use by subjects (both pediatric subjects and caregivers) considered representative of the intended user population.
  • all injections will be performed in a room designed to represent a home-like setting for patients and caregivers. Prescribing a self-injectable to pediatrics is a serious matter to the health care community and training is always provided as a requirement before caregivers can inject their children at home or to allow patients to self-inject.
  • the device and IFU was developed within this context. Furthermore, the Sponsor has evaluated the injection naive patient population in formative testing. Based on this testing, the VARIOJECT® (injector) is considered reasonably intuitive to users. The Sponsor has taken steps to improve the design based on prior formative testing. As an example, formative testing has shown that untrained users are inclined to skip steps related to priming (ensuring proper device orientation and tapping bubbles to the top).
  • the Sponsor has incorporated colored graphics on the device and clear step-by-step instructions. Although there has been significant focus and effort to improve the usability of the device and associated IFU, the required training has proven to be the best way to ensure proper use. As such, for the human factors study, all patients and family caregivers will be provided with representative, though minimal, training thus mimicking the real-world context in which these devices will be deployed. The Sponsor views the simulated human factors study as pivotal to demonstrating that the device is safe for in-home-use and is sufficient to address the clinical risks associated with this device.
  • VARIOJECT® injector
  • pediatric UC patients designed to capture and document real-life VARIOJECT® (injector) handling and use experience data from injections administered by subjects and caregivers in the home-setting, including complaints and failures in use.
  • the actual use study is considered as additional support of the pivotal human factors data.
  • a 4.5mm needle length was chosen for VARIOJECT® (injector) based on published literature 1 ⁇ 2 pertaining to needle lengths for injecting subcutaneously in children. This needle length was chosen to account for both manufacturing tolerances on glass syringes as well as tolerances associated with assembly of the syringe in the VARIOJECT® (injector) in order to appropriately balance the small risks of intramuscular and intradermal delivery. Based on data provided in the published literature, the risk of intramuscular injections in the 7-17 year age group is low, and although the risk of intramuscular injections is slightly higher for the 2-6 year age group, the risk is still low.
  • mAbs do not appear to require precise or device- specific administration into a particular location in the subcutaneous space to be safe and effective, and small variations in PK (although not expected) are not likely to impact efficacy outcomes. Nonetheless, safety assessments will be collected as part of the proposed study.
  • T1D progression of T1D is associated with a significant impact on quality of life in early stages and eventual morbidity and mortality.
  • a number of compounds have been explored in those newly diagnosed with T1D to maintain residual beta cell function, which in turn will improve glycemic control and reduce short- and long-term complications of disease.
  • the Sponsor believes there is an unmet medical need and important opportunity to delay or prevent T1D in those who are at high risk. It is well established that the autoimmune-mediated b-cell loss, was initiated many years before the clinical diagnosis.
  • autoimmune process that is occurring at the time of clinical diagnosis, and thus being targeted in “new onset” studies, is likely very similar to that which had been occurring in the preceding months or years, and thus agents that show (even modest) efficacy in newly diagnosed T1D can be considered as candidates to examine in those at high risk of developing T1D (i.e., “Pre-TID”).
  • Pre-TID This is evidenced through the support/approval of the Agency for studies of abatacept (NCT01773707) and teplizumab (NCT01030861) in those with serologic evidence of T1D autoimmunity but has not yet met diabetes mellitus clinical criteria.
  • treating earlier to intercept or delay onset of disease may have both near and long-term benefits.
  • young children/adolescents may avoid the requirement of multiple daily injections of exogenous insulin.
  • patients diagnosed with T1D at younger ages may have more aggressive disease. Therefore, delaying onset by 2 or more years may avoid more rapid progression of b-cell destruction.
  • early preservation of b-cell mass and good glycemic control may mitigate severe complications later in life such as cardiovascular disease or hospitalization which are associated with significant morbidity and mortality, and socioeconomic burden.
  • the Sponsor considers that children/adolescent patients 6 to 21 years of age with >2 auto-antibodies for T1D with dysglycemia who are not yet insulin dependent are patients greatest at-risk for progression to clinical disease and would most benefit from treatment in the pre-disease state.
  • the Sponsor proposes to take a staged approach in determining whether golimumab is an effective treatment that provides benefit in the target population described above.
  • the Sponsor’s first step in the staged approach is to establish the benefit-risk of golimumab in new onset T1D study described in this Briefing Document.
  • the results of this study provide key information on the effect of golimumab on preservation of b-cell mass as well as the safety profile in this patient population.
  • the goal of this pilot study was to obtain early safety and efficacy information to establish the mechanism of benefit with golimumab in the pre-disease state.
  • the Sponsor intends to communicate the results from these two initial studies in the context of preparations for the proof-of-concept study in those at risk for T1D to align with the Agency on a robust development plan for golimumab in the treatment of pre-TID.
  • FDA Food and Drug Administration
  • HCP Health Care Professional
  • HFS Human Factors Study
  • IFU Instructions for use
  • PFS prefilled syringe
  • PFS- U prefilled syringe in the ULTRASAFE PASSIVE® (needle guard)
  • PK pharmacokinetic(s)
  • sBLA Supplemental Biologies License Application
  • UC ulcerative colitis.
  • VARIOJECT® (injector) Actual Use Study (includes assessment of labeling comprehension, safety, and device durability/robustness). This study will be conducted as a substudy of CNT0148UC01002 which will be conducted to provide PK data in pediatric with body weight ⁇ 45 kg to support an extrapolation- based approach to the pediatric UC indication, and to demonstrate that the VARIOJECT® (injector) device can achieve the expected drug exposure in the intended pediatric population.
  • VARIOJECT® injector
  • the VARIOJECT® injector
  • the appropriate training and written Instructions for Use is suitable for at- home administration by subjects or their caregivers.
  • Study and questionnaire were designed to capture and document real-life PFS-U handling and use experience data from injections administered by subjects and caregivers in the home-setting, including complaints and failures in use.
  • the study assigns pass/fail criteria at the individual task level, and behaviors such as errors, close calls, and/or difficulties were recorded at the individual task level.
  • Subjective participant feedback was collected in narrative form, and participants were asked open-ended questions about the procedure and device design. Participants were asked probe questions to evaluate their knowledge and understanding of the instructions provided in the IFU.
  • Verification testing is conducted according to the following.
  • VARIOJECT® injector
  • D2 - integrated, single dose, non-replaceable container whereby a portion of the deliverable volume is expelled.
  • Functional stability testing evaluates aging of the drug-device combination product followed by device testing to ensure device functionality.
  • the assembled product is stored at the recommended temperature of 2-8 °C, as well as accelerated (25 °C) conditions.
  • Device sub-assemblies are exposed to elevated temperature aging followed by assembly with the drug-filled PFS and device testing to ensure device functionality.
  • the accelerated aging data is supported by real time aging testing of device sub-assemblies stored at room temperature.
  • Assembly process validation involves assembly of drug-device combination product using equipment that will be used to assemble the VARIOJECT® (injector) for commercial launch, followed by device testing.
  • the simulated use (safety) study will include 500+ mock injections demonstrating successful operation of needle safety feature following ISO 23908:2011 Sharps injury protection — Requirements and test methods - Sharps protection features for single-use hypodermic needles, introducers for catheters and needles used for blood sampling and Guidance for Industry and FDA Staff: Medical Devices with Sharps Injury Prevention Features.
  • Biocompatibility testing is performed in accordance with ISO-10993-1 : 2009 Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk management process for skin contacting surface device having limited contact duration ( ⁇ 24 hours).
  • Table 8 Dose chart for study CNT0148DML2001 in pediatric subjects with Type 1 Diabetes with body weight ⁇ 45
  • SIMPONI ® golimumab
  • DPARP Pulmonary, Allergy, and Rheumatology Products
  • DGIEP Gastroenterology and In-Bom Error Products
  • SIMPONI ® in combination with methotrexate, is indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis.
  • SIMPONI ® (golimumab), alone or in combination with methotrexate, is indicated for the treatment of adult patients with active psoriatic arthritis.
  • SIMPONI ® (golimumab) is indicated for the treatment of adult patients with active ankylosing spondylitis.
  • SIMPONI ® (golimumab) is indicated in adult patients with moderately to severely active ulcerative colitis who have demonstrated corticosteroid dependence or who have had an inadequate response to or failed to tolerate oral aminosalicylates, oral corticosteroids, azathioprine, or 6-mercaptopurine for:
  • the Sponsor has 4 active INDs in support of the golimumab development program with DPARP or DGIEP
  • the Sponsor proposes to either submit items to the new IND or cross-refer to IND 09925 or BLA125289. These will be text cross-references only (no electronic hyperlinks).
  • Table 9 IND requirements Table Topline Results for Phase 2a Clinical Study - Treatment with SIMPONI® (golimumab) to arrest loss of b-cell function in Type 1 Diabetes (T1D)
  • CNT0148DML2001 is an ongoing Phase 2a, proof-of-concept, multicenter, randomized, double-blind, placebo-controlled study of the efficacy and safety of golimumab compared with placebo in subjects 6 through 21 years old who have been recently diagnosed with T1D. This study in a newly diagnosed population is also an initial step in the development program to better understand how TNF inhibition can provide benefits in pre-TID and other T1D patients by stabilizing endogenous insulin production and preventing or delaying disease progression.
  • the target population must present a peak stimulated C-peptide level >0.2 pmol/mL following a 4-hour Mixed Meal Tolerance Test (MMTT) obtained at study screening and be positive for at least 1 of the following diabetes-related autoantibodies: GAD-65, IA-2, ZnT8, ICA, or insulin.
  • Subjects must be randomized within 100 days after diagnosis of T1D.
  • Eligible subjects were randomized in a 2: 1 ratio to golimumab or placebo using a permuted-block randomization procedure stratified by baseline 4-hour C-peptide AUC ( ⁇ 0.66 pmol/mL, or >0.66 pmol/mL)
  • Treatment duration/Trial duration Treatment duration/Trial duration:
  • the duration of study participation is 108 weeks, including a 4-week screening period, a 52-week treatment followed by a 52-week off-therapy follow-up period.
  • Optional Extension Period Patients in the study meeting response criteria at Week 52, may enter in an open-label (OL) extension period to receive golimumab SC for an additional period of time.
  • This additional period of time, or extension period can be the same as the initial period of treatment or any time period as needed to maintain or to improve the condition of the patient.
  • the extension periods can be referred to as cycles or repeated time periods of treatment to maintain or to improve the condition of the patient.
  • the extension period may also be referred to as a maintenance period or period of time when the patient receives a maintenance dose.
  • the maintenance dose can be the same dose or a different dose than used in the original time period of the study.
  • the sample size calculation was based on the primary endpoint, an MMTT 4- hour C-peptide AUC at Week 52. Due to skewed C-peptide AUC data, normalizing transformation of log (AUC +1) was applied for sample size assessment. Based on published external data, a common standard deviation (SD) of log (AUC+1) of 0.215 was assumed, and the back-transformed means for 4-hour C-peptide AUC were assumed to be 0.385 and 0.635 for the placebo and golimumab groups respectively, i.e., the expected treatment difference (back-transformed) was 0.25. Based on these assumptions, with 81 subjects (54 on golimumab and 27 on placebo), there was 90% power to detect the treatment difference with a 2-sided significance level of 0.05 by using a 2-sample t-test.
  • SD standard deviation
  • golimumab can preserve b-cell function in children and young adults with newly diagnosed T1D.
  • This topline result summary is specific to the information collected through Week 52 (primary endpoint) database lock (July 3 rd , 2019).
  • MMTT stimulated 4-hour C-peptide AUC data was analyzed in log(AUC+l) scale.
  • the LSMean change from baseline at Week 52 was -0.27 and -0.10 for the placebo and golimumab groups, respectively (FIG. 25).
  • the insulin usage substantially increased from baseline in the placebo group and slightly increased in the golimumab group: the LSMean changes were 0.244 and 0.066 units/kg/day for the placebo and golimumab groups, respectively.
  • Safety Table 11 summarizes key safety events. Through Week 52, among all treated subjects:
  • hypoglycemia events could also be reported as AEs; AEs of hypoglycemia were reported in 23.2% versus 7.1% of participants with golimumab and placebo, respectively, with reports from 6/27 sites and 15/84 participants, including in all 9 participants from a single site.
  • the severity of hypoglycemia adverse events was not different from cases of hypoglycemia not reported as adverse events, with no cases of severe hypoglycemia reported.
  • CCAE Common Terminology Criteria for Adverse Events
  • Week 52 assessments The nine participants who withdrew from the study and did not have assessments at Week 52 were considered non-responders.
  • the objective was to evaluate pharmacokinetic (PK) and pharmacodynamic (PD) data from this TIGER study by developing a population pharmacokinetic (PopPK) model and performing exposure-response (ER) analyses.
  • the PopPK model was developed using data from this TIGER study and two other pediatric studies with SC golimumab (PURSUIT-PEDS-PK and GO-KIDS).
  • ER analyses were conducted for change from baseline in C-peptide AUC (primary endpoint) and HbAlc (secondary endpoint) levels at Weeks 12, 26 and 52.
  • the developed PopPK model was able to adequately describe the observed PK of golimumab in T1DM patients. Body weight and immune response to treatment were covariates with significant effects. Golimumab treatment showed significant treatment benefit in patients with newly diagnosed T1DM with changes in C-peptide AUC as the primary endpoint when active treatment group is compared with placebo treatment group. ER analyses did not show clear trends within the active treatment group which may indicate that the exposure from this TlDM-specific dosing regimen was at the plateau of the ER curve.
  • the primary endpoint is the MMTT-stimulated 4-hour C-peptide AUC (pmol/mL) at Week 52. Due to the skewed distribution of C-peptide AUC data, a normalization transformation of log (AUC+1) was applied.
  • a Mixed Model Repeated Measure (MMRM) model was utilized to assess the primary efficacy endpoint, in which post-baseline log(AUC+l) was the response variable. The model was adjusted for baseline AUC, age, gender, time, baseline-by-time interaction, and treatment-by time interaction.
  • the mean (SD) MMTT-stimulated 4-hour C-peptide AUC (pmol/mU) was 0.64 (0.423) for the golimumab treatment group and 0.43 (0.388) for the placebo group, with a p-value of 0.0004 after adjusting for baseline and other pre-specified factors, indicating that the decline in C-peptide AUC was less in the golimumab treatment group versus placebo, thereby successfully meeting the primary objective of the study.
  • Table 14 Summary of MMTT-stimulated 4 hour C peptide AUC (pmol/mL) at Week 52 (Primary Analysis); Full Analysis Set.
  • a normalization transformation of log (A uc +i) was applied.
  • the back-transformation is exp(estimate) -1, where the estimate was based on log (A uc +i) .
  • b MMRM was applied with post-baseline log (A uc +i) as the response variable, gender, treatment, time, and treatment-by-time interaction as categorical effects, as well as baseline, baseline-by-time, and age as covariates.
  • Table 15 Forest Plot of Change From Baseline in log(AUC+l) at Week 52 for Subgroups Defined by Baseline Disease Characteristics; Full Analysis Set
  • the secondary endpoints are:
  • Hypoglycemic event rates (defined as blood glucose levels of ⁇ 70mg/dL or events reported by subjects in the absence of a BG reading) through Week 52. 0
  • Each continuous secondary endpoint (insulin use and HbAlc) was analyzed using an MMRM model.
  • the model was adjusted for baseline, age, gender, time, baseline-by-time, and treatment-by-time interactions.
  • the number of hypoglycemia events was analyzed using a Poisson regression model, accounting for the duration of study participation through week 52.
  • the model was adjusted for baseline HbAlc and age.
  • R Responder partial remission: subjects in partial remission, which was defined as an insulin dose-adjusted Ale (IDAAlc) of ⁇ 9.
  • the IDAAlc is calculated as follows: HbAlc (%) + (4 c insulin dose [U/kg/day]).
  • IDAAlc ⁇ 9 is a definition of partial remission that includes both glycemic control and insulin dose. It reflects residual b-cell function and may have better stability compared with other conventional definitions (Mortensen, et al. Diabetes Care 2009;32:1384-90).
  • FIG. 34 shows proportion (%) of subjects in partial remission of T1D over time for treatment with golimumab and placebo.
  • hypoglycemia was defined as a biochemically confirmed blood glucose reading of ⁇ 70 mg/dL irrespective of clinical symptoms or sequelae and all events of severe hypoglycemia, denoting reported as such and per current recommendations as Level 1 (blood glucose reading of 54 to ⁇ 70 mg/dL), Level 2 (blood glucose reading of ⁇ 54 mg/dL), or Level 3 (severe cognitive impairment requiring external assistance for recovery.
  • This subpopulation analysis of patients aged 6-17 ( ⁇ 18 years old) shows that children treated with golimumab have a number substantial metabolic and clinical benefits compared to patients treated with placebo. Thus, golimumab can be considered as potential disease modifying therapy for this population.
  • Table 17 Comparison of Insulin Usage at Week 52 and Hypoglycemic Events Through Week 52
  • the annual hypoglycemia event rates were 39.01 and 43.36 in the golimumab and placebo treatment groups, respectively. Compared to the placebo group, a 10% reduction in annual hypoglycemia event rate was observed in the golimumab treatment group. There was no severe hypoglycemia event reported through Week 52
  • T1D is a disease associated with significant morbidity and clinical complications with continued deterioration of beta-cell function.
  • the Sponsor is currently conducting CNT0148DML2001, a Phase 2a randomized, double-blind, placebo-controlled, parallel-group, multicenter, study of golimumab in subjects with new onset T1D (Stage 3). The purpose of this ongoing study is to evaluate whether subcutaneously administered golimumab can maintain residual b-cell function and improve metabolic control in children and young adults with newly diagnosed T1D.
  • the current study in newly diagnosed patients has enrolled male or female subjects 6 through 21 years of age who meet the American Diabetes Association (ADA) standard T1D criteria within 100 days of randomization.
  • subjects must have a stimulated C-peptide level >0.2 pmol/mL following a 4-hour MMTT obtained at study screening and have been positive for at least 1 of the following diabetes-related autoantibodies obtained at study screening:
  • IA-2 insulinoma-associated antigen-2
  • ICA islet cell antigen
  • Insulin if obtained within 10 days of the onset of exogenous insulin therapy
  • SIMPONI® (golimumab) preserves beta-cell function
  • beta-cell is a highly secretory endocrine cell tasked with a heavy biosynthetic burden that requires a robust functional endoplasmic reticulum (ER) for efficient translation and processing to produce insulin.
  • ER endoplasmic reticulum
  • insulin demand can exceed the ability of the ERto process newly translated proteins and lead to ER stress.
  • This ER stress can act to initiate or augment autoimmune-mediated beta-cell dysfunction and pro-apoptotic signaling pathways that can ultimately lead to beta-cell death (Mirmira et al., Curr Diab Rep.
  • the proinsulin-to-C-peptide ratio is used as an indicator of how efficiently the ER in beta- cells are processing proinsulin to insulin and serves as a biomarker for ER stress and associated beta-cell stress that affects beta-cell-function (Sims et al., Diabetes Care 2016;39: 1519-1526).
  • interventions that preserve beta-cell function by reducing beta-cell stress as measured by the proinsulin-to-C-peptide ratio offer the promise of more effective treatments and even prevention of T1D.
  • Fasting total proinsulin levels can also be determined, e.g., with a STELLUX® (Chemi Human Total Proinsulin ELISA kit) (ALPCO, Salem, NH).
  • Fasting C-peptide levels can also be determined, e.g., with an ACCUBIND® (ELISA kit) (Monobind Inc., Lake Forest, CA).
  • the proinsulin-to-C-peptide ratios are calculated as the molar ratio of proinsulin concentrations to C-peptide concentrations and the molar ratios can be multiplied by 100% to get proinsulin concentrations as a percentage of C-peptide concentrations.
  • the threshold for the current study was stimulated C-peptide level >0.2 pmol/mL following a 4-hour Mixed Meal Tolerance Test (MMTT), but treatment of subjects with stimulated C-peptide concentration >0.2 nmol/mL and even lower levels of C-peptide could be appropriate and is supported by the results from this study and results reported in Lachin et al., Diabetes. 2014 Feb; 63(2): 739-74.
  • Lachin et al indicated that preservation of stimulated C-peptide levels of >0.2 nmol/L had meaningful clinical outcomes, but so also did an increase in concentration of C- peptide across a range of values.
  • Golimumab resulted in -41% of participants having increases or ⁇ 5% reductions in C-peptide AUC overtime. Moreover, golimumab was associated with increases and longer times in partial remission (known as the “honeymoon period”), as determined by the IDAAlc score, which includes both insulin use and HbAlc. This clinically relevant period is characterized by optimal glycemic control with low insulin doses (Mortensen et al. Diabetes Care 2009;32:1384-90). Additionally, support of improved b-cell health is suggested by the proinsulin/C -peptide ratio, an established measure of b-cell stress and dysfunction (Mirmira, et al. CurrDiab Rep 2016;16:95), which was largely unchanged in golimumab recipients versus increased in placebo recipients.
  • TNFa in T1D has not been clear from preclinical studies, but findings of this study are consistent with those of a small Phase 1, placebo-controlled trial study investigating the TNFa fusion protein, etanercept, in children aged 3-18 years with new-onset T1D for 24 weeks. Over 6 months, this study also found that there were improvements in C-peptide AUC and a reduction in insulin usage with TNFa-blockade (P ⁇ 0.05).
  • TNFa blockers are approved in some autoimmune conditions for children as young as 2 years of age (eg, juvenile idiopathic arthritis and inflammatory bowel disease) and collectively have a well-established benefit: risk profile.
  • TNFa blockers including golimumab
  • golimumab are used to treat childhood diseases that have similar disease burdens as T1D and many of these other diagnoses require chronic therapy with golimumab to maintain remission.
  • Additional studies are necessary to evaluate the treatment regimen needed to prolong endogenous insulin production beyond 52 weeks. However, few if any other autoimmune conditions are managed with a single course of immunomodulatory therapy and it is likely that additional courses of therapy will be needed to achieve durable, long lasting effects. Additional analyses are ongoing to determine if subpopulations in this study were differentially responsive to golimumab.
  • combination therapy with other immune modulators, b-cell protective therapies and/or b-cell replacement may lead to improved efficacy.
  • Example 10 Preserving beta-cell function in a human patient
  • a method to preserve beta-cell function in a human patient is achieved by administering a TNF inhibitor, as follows. There is selection of a human patient in need of treatment to preserve beta-cell function. Determination is made of the fasting proinsulin-to-C-peptide ratio of the patient before administering the TNF inhibitor to the patient. Administering, at a selected dose, the TNF inhibitor to the patient. Next, the fasting proinsulin-to-C-peptide ratio of the patient, after administering the TNF inhibitor, is determined. Comparison of the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor is performed to determine if there is a change in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor to the patient.
  • T1D Type 1 Diabetes
  • pre-TID pre-Type 1 Diabetes
  • Example 11 Preserving beta-cell function in a human patient
  • a method to preserve beta-cell function in a human patient is achieved by administering a TNF inhibitor, as follows. There is selection of a human patient in need of treatment to preserve beta-cell function. Determination is made of the fasting proinsulin-to-C-peptide ratio of the patient before administering the TNF inhibitor to the patient. Administering, at a selected dose, the TNF inhibitor to the patient wherein the inhibitor is Simponi. Next, the fasting proinsulin-to-C-peptide ratio of the patient, after administering the TNF inhibitor, is determined.
  • Comparison of the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor is performed to determine if there is a change in the fasting proinsulin-to-C- peptide ratio after administering the TNF inhibitor to the patient.
  • a determination is made of beta-cell function preservation in the patient to determine if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or if there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor.
  • Example 12 Preserving beta-cell function in a human patient
  • a method to preserve beta-cell function in a human patient is achieved by administering a TNF inhibitor, as follows. There is selection of a human patient in need of treatment to preserve beta-cell function. Determination is made of the fasting proinsulin-to-C-peptide ratio of the patient before administering the TNF inhibitor to the patient. Administering, at a selected dose, the TNF inhibitor to the patient wherein the inhibitor is Remicade. Next, the fasting proinsulin-to-C-peptide ratio of the patient, after administering the TNF inhibitor, is determined.
  • Comparison of the fasting proinsulin-to-C-peptide ratio determined before and after administering the TNF inhibitor is performed to determine if there is a change in the fasting proinsulin-to-C- peptide ratio after administering the TNF inhibitor to the patient.
  • a determination is made of beta-cell function preservation in the patient to determine if there is not an increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor or if there is a minimal increase in the fasting proinsulin-to-C-peptide ratio after administering the TNF inhibitor.

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Abstract

L'invention concerne des matériaux et des méthodes de traitement du diabète de type 1 (T1D) avec un inhibiteur de TNF, par exemple, l'inhibiteur de TNF étant l'anticorps anti-TNF SIMPONI ® (gomumab) ayant une chaîne lourde (HC) comprenant SEQ ID NO : 36 et une chaîne légère (LC) comprenant SEQ ID NO : 37 ou un fragment de liaison à l'antigène de celui-ci.
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Citations (145)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
EP0003089A1 (fr) 1978-01-06 1979-07-25 Bernard David Séchoir pour feuilles imprimées par sérigraphie
US4239754A (en) 1976-10-23 1980-12-16 Choay, S.A. Liposomes containing heparin and a process for obtaining them
US4309989A (en) 1976-02-09 1982-01-12 The Curators Of The University Of Missouri Topical application of medication by ultrasound with coupling agent
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1986005803A1 (fr) 1985-03-30 1986-10-09 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4656134A (en) 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
EP0218868A2 (fr) 1985-08-29 1987-04-22 New York Blood Center, Inc. Préparation de facteur nécrotique tumoral humain pur et hybridomes produisant des anticorps monoclonaux contre le facteur nécrotique tumoral humain
US4668218A (en) 1985-04-12 1987-05-26 Aktiebolaget Draco Indicating means for a dosage dispensing device
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
EP0237507A1 (fr) 1986-03-07 1987-09-16 Aktiebolaget Draco Dispositif d'inhalation de poudre
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US4766067A (en) 1985-05-31 1988-08-23 President And Fellows Of Harvard College Gene amplification
US4767402A (en) 1986-07-08 1988-08-30 Massachusetts Institute Of Technology Ultrasound enhancement of transdermal drug delivery
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
EP0288088A2 (fr) 1987-04-24 1988-10-26 Teijin Limited Détection du facteur nécrosant de tumeur; anticorps monoclonal et composition
US4795699A (en) 1987-01-14 1989-01-03 President And Fellows Of Harvard College T7 DNA polymerase
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1989006283A1 (fr) 1988-01-11 1989-07-13 Ingene (International Genetic Engineering, Inc.) Nouveau vecteur de plasmide avec sequence a fonction de signal pour pectate lyase
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
WO1990004036A1 (fr) 1988-10-12 1990-04-19 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
WO1990003809A1 (fr) 1988-10-12 1990-04-19 Rochal Industries, Inc. Bandage conforme et matiere d'enduction
US4921794A (en) 1987-01-14 1990-05-01 President And Fellows Of Harvard College T7 DNA polymerase
US4925673A (en) 1986-08-18 1990-05-15 Clinical Technologies Associates, Inc. Delivery systems for pharmacological agents encapsulated with proteinoids
EP0368684A1 (fr) 1988-11-11 1990-05-16 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
EP0371998A1 (fr) 1987-07-24 1990-06-13 Int Genetic Eng Ensemble modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
US4939666A (en) 1987-09-02 1990-07-03 Genex Corporation Incremental macromolecule construction methods
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4956288A (en) 1988-04-22 1990-09-11 Biogen, Inc. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
WO1990014424A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Procede d'isolement de recepteurs presentant une specificite preselectionnee
WO1990014430A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique
WO1990014443A1 (fr) 1989-05-16 1990-11-29 Huse William D Coexpression de recepteurs heteromeres
WO1991000360A1 (fr) 1989-06-29 1991-01-10 Medarex, Inc. Reactifs bispecifiques pour le traitement du sida
US4994370A (en) 1989-01-03 1991-02-19 The United States Of America As Represented By The Department Of Health And Human Services DNA amplification technique
WO1991002078A1 (fr) 1989-08-07 1991-02-21 Peptide Technology Ltd Ligands de liaison du facteur de necrose de tumeurs
US5066584A (en) 1988-09-23 1991-11-19 Cetus Corporation Methods for generating single stranded dna by the polymerase chain reaction
WO1992000373A1 (fr) 1990-06-29 1992-01-09 Biosource Genetics Corporation Production de melanines a l'aide de microorganismes transformes
US5091310A (en) 1988-09-23 1992-02-25 Cetus Corporation Structure-independent dna amplification by the polymerase chain reaction
WO1992007076A1 (fr) 1990-10-18 1992-04-30 The Mathilda And Terence Kennedy Institute Of Rheumatology Recepteur de tnfalpha (facteur de necrose tumorale alpha) humain modifie
US5116964A (en) 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5122464A (en) 1986-01-23 1992-06-16 Celltech Limited, A British Company Method for dominant selection in eucaryotic cells
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5142033A (en) 1988-09-23 1992-08-25 Hoffmann-La Roche Inc. Structure-independent DNA amplification by the polymerase chain reaction
US5149636A (en) 1982-03-15 1992-09-22 Trustees Of Columbia University In The City Of New York Method for introducing cloned, amplifiable genes into eucaryotic cells and for producing proteinaceous products
WO1992016221A1 (fr) 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5225538A (en) 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
US5231024A (en) 1986-09-13 1993-07-27 Basf Aktiengesellschaft Monoclonal antibodies against human tumor necrosis factor (tnf), and use thereof
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5266491A (en) 1989-03-14 1993-11-30 Mochida Pharmaceutical Co., Ltd. DNA fragment and expression plasmid containing the DNA fragment
WO1994006498A1 (fr) 1992-09-23 1994-03-31 Fisons Plc Inhalateur
US5304489A (en) 1987-02-17 1994-04-19 Genpharm International, Inc. DNA sequences to target proteins to the mammary gland for efficient secretion
WO1994008552A2 (fr) 1992-10-19 1994-04-28 Dura Pharmaceuticals, Inc. Inhalateur pour poudre seche
GB2272440A (en) 1990-08-29 1994-05-18 Genpharm Int Transgenic non-human animals capable of producing heterologous antibodies
WO1994016970A1 (fr) 1993-01-19 1994-08-04 Glaxo Group Limited Dispositif
EP0614989A1 (fr) 1993-02-17 1994-09-14 MorphoSys AG Procédé de sélection in vivo des protéines attachées des ligands
WO1994025585A1 (fr) 1993-04-26 1994-11-10 Genpharm International, Inc. Animaux transgeniques capables de produire des anticorps heterologues
US5404871A (en) 1991-03-05 1995-04-11 Aradigm Delivery of aerosol medications for inspiration
WO1995016027A1 (fr) 1993-12-06 1995-06-15 Bioinvent International Ab Procede de selection de bacteriophages specifiques
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US5447851A (en) 1992-04-02 1995-09-05 Board Of Regents, The University Of Texas System DNA encoding a chimeric polypeptide comprising the extracellular domain of TNF receptor fused to IgG, vectors, and host cells
US5458135A (en) 1991-07-02 1995-10-17 Inhale Therapeutic Systems Method and device for delivering aerosolized medicaments
US5496549A (en) 1990-04-02 1996-03-05 Takeda Chemical Industries, Ltd. Bispecific monoclonal antibodies, thrombolytic agent and method of cell lysis
WO1996007754A1 (fr) 1994-09-02 1996-03-14 The Scripps Research Institute Procedes de production de banques d'anticorps au moyen de chaines legeres d'immunoglobuline universelles ou rendues alleatoires
US5514670A (en) 1993-08-13 1996-05-07 Pharmos Corporation Submicron emulsions for delivery of peptides
EP0710719A1 (fr) 1990-01-12 1996-05-08 Cell Genesys, Inc. Génération d'anticorps xénogemiques
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
WO1996034096A1 (fr) 1995-04-28 1996-10-31 Abgenix, Inc. Anticorps humains derives de xeno-souris immunisees
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5580734A (en) 1990-07-13 1996-12-03 Transkaryotic Therapies, Inc. Method of producing a physical map contigous DNA sequences
US5582996A (en) 1990-12-04 1996-12-10 The Wistar Institute Of Anatomy & Biology Bifunctional antibodies and method of preparing same
US5595898A (en) 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5601819A (en) 1988-08-11 1997-02-11 The General Hospital Corporation Bispecific antibodies for selective immune regulation and for selective immune cell binding
WO1997013852A1 (fr) 1995-10-10 1997-04-17 Genpharm International, Inc. Animaux non humains transgeniques pouvant produire des anticorps heterologues
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5625825A (en) 1993-10-21 1997-04-29 Lsi Logic Corporation Random number generating apparatus for an interface unit of a carrier sense with multiple access and collision detect (CSMA/CD) ethernet data network
US5627052A (en) 1990-08-02 1997-05-06 B.R. Centre, Ltd. Methods for the production of proteins with a desired function
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5641670A (en) 1991-11-05 1997-06-24 Transkaryotic Therapies, Inc. Protein production and protein delivery
WO1997022376A1 (fr) 1995-12-20 1997-06-26 Intersurgical Limited Nebuliseur
US5643768A (en) 1989-10-05 1997-07-01 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5643759A (en) 1993-10-30 1997-07-01 Biotest Pharma Gmbh Method for preparing bispecific monoclonal antibodies
WO1997025086A2 (fr) 1996-01-03 1997-07-17 Glaxo Group Limited Dispositif d'inhalation
US5656730A (en) 1995-04-07 1997-08-12 Enzon, Inc. Stabilized monomeric protein compositions
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
EP0814259A1 (fr) 1996-06-19 1997-12-29 Motorenfabrik Hatz GmbH & Co. KG Dispositif de démarrage à froid
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
US5733761A (en) 1991-11-05 1998-03-31 Transkaryotic Therapies, Inc. Protein production and protein delivery
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5763733A (en) 1994-10-13 1998-06-09 Enzon, Inc. Antigen-binding fusion proteins
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
WO1998024884A1 (fr) 1996-12-02 1998-06-11 Genpharm International Animaux transgeniques non humains capables de produire des anticorps heterologues
WO1998024893A2 (fr) 1996-12-03 1998-06-11 Abgenix, Inc. MAMMIFERES TRANSGENIQUES POSSEDANT DES LOCI DE GENES D'IMMUNOGLOBULINE D'ORIGINE HUMAINE, DOTES DE REGIONS VH ET Vλ, ET ANTICORPS PRODUITS A PARTIR DE TELS MAMMIFERES
US5766886A (en) 1991-12-13 1998-06-16 Xoma Corporation Modified antibody variable domains
US5770428A (en) 1993-02-17 1998-06-23 Wisconsin Alumni Research Foundation Chimeric retrovial expression vectors and particles containing a simple retroviral long terminal repeat, BLV or HIV coding regions and cis-acting regulatory sequences, and an RNA translational enhancer with internal ribsome entry site
US5770222A (en) 1989-12-22 1998-06-23 Imarx Pharmaceutical Corp. Therapeutic drug delivery systems
US5770198A (en) 1988-05-18 1998-06-23 The Research Foundation Of The State Of New York Platelet-specific chimeric 7E3 immunoglobulin
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
WO1998035888A1 (fr) 1997-02-13 1998-08-20 Glaxo Group Limited Valve doseuse pour aerosols
US5807706A (en) 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US5814599A (en) 1995-08-04 1998-09-29 Massachusetts Insitiute Of Technology Transdermal delivery of encapsulated drugs
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5833985A (en) 1994-03-07 1998-11-10 Medarex, Inc. Bispecific molecules for use in inducing antibody dependent effector cell-mediated cytotoxicity
WO1998050433A2 (fr) 1997-05-05 1998-11-12 Abgenix, Inc. Anticorps monoclonaux humains contre le recepteur du facteur de croissance epidermique
US5839446A (en) 1992-10-28 1998-11-24 Transmedica International, Inc. Laser perforator
WO1998053847A1 (fr) 1997-05-29 1998-12-03 Ben Gurion University Of The Negev Research And Development Authority Systeme de transport transdermique
US5849695A (en) 1993-01-13 1998-12-15 The Regents Of The University Of California Parathyroid hormone analogues useful for treatment of osteoporosis and disorders of calcium meatabolism in mammals
US5851198A (en) 1995-10-10 1998-12-22 Visionary Medical Products Corporation Gas pressured needle-less injection device and method
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
US5871753A (en) 1993-02-12 1999-02-16 Board Of Trustees Of The Leland Stanford Junior University Regulated transcription of targeted genes and other biological events
US5879681A (en) 1997-02-07 1999-03-09 Emisphere Technolgies Inc. Compounds and compositions for delivering active agents
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US5959084A (en) 1990-10-29 1999-09-28 Chiron Corporation Bispecific antibodies, methods of production and uses thereof
US5959083A (en) 1991-06-03 1999-09-28 Behringwerke Aktiengellschaft Tetravalent bispecific receptors, the preparation and use thereof
US5989530A (en) 1992-03-10 1999-11-23 Goldwell Ag Bleaching composition for human hair and process for its production
US6010902A (en) 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
US6037453A (en) 1995-03-15 2000-03-14 Genentech, Inc. Immunoglobulin variants
US6060285A (en) 1989-03-23 2000-05-09 Roche Diagnostics Gmbh Process for the production of hetero-bispecific antibodies
US6132992A (en) 1993-02-01 2000-10-17 Bristol-Myers Squibb Co. Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US6193967B1 (en) 1992-10-02 2001-02-27 Peter M. Morganelli Bispecific reagents for redirected targeting of human lipoproteins
US6210668B1 (en) 1996-09-03 2001-04-03 Gsf Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Destruction of contaminating tumor cells in stem cell transplants using bispecific antibodies
WO2002012500A2 (fr) 2000-08-07 2002-02-14 Centocor, Inc. Anticorps, compositions, procedes anti-il-12 et leur utilisation
US7250165B2 (en) 2000-08-07 2007-07-31 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US7815909B2 (en) 2000-08-07 2010-10-19 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US9107149B2 (en) 2010-04-07 2015-08-11 Lenovo (Singapore) Pte Ltd Wireless portable computer capable of autonomously adjusting load of wireless base station
US9105939B2 (en) 2008-03-10 2015-08-11 Nissan Motor Co., Ltd. Battery with battery electrode and method of manufacturing same
US9102989B2 (en) 2002-05-23 2015-08-11 Cognis Ip Management Gmbh Non-revertible β-oxidation blocked Candida tropicalis
US9109630B2 (en) 2013-01-15 2015-08-18 Jtekt Corporation Rolling bearing unit
US9401234B2 (en) 2013-03-22 2016-07-26 Polytronics Technology Corp. Over-current protection device
US9618978B2 (en) 2013-10-03 2017-04-11 Acer Incorporated Methods for controlling a touch panel and portable computers using the same
WO2017136524A2 (fr) * 2016-02-05 2017-08-10 Janssen Biotech, Inc. Anticorps anti-tnf, compositions, méthodes et utilisation pour le traitement ou la prévention du diabète de type 1
US9816280B1 (en) 2016-11-02 2017-11-14 Matthew Reitnauer Portable floor

Patent Citations (192)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4309989A (en) 1976-02-09 1982-01-12 The Curators Of The University Of Missouri Topical application of medication by ultrasound with coupling agent
US4239754A (en) 1976-10-23 1980-12-16 Choay, S.A. Liposomes containing heparin and a process for obtaining them
EP0003089A1 (fr) 1978-01-06 1979-07-25 Bernard David Séchoir pour feuilles imprimées par sérigraphie
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4656134A (en) 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
US5149636A (en) 1982-03-15 1992-09-22 Trustees Of Columbia University In The City Of New York Method for introducing cloned, amplifiable genes into eucaryotic cells and for producing proteinaceous products
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5385839A (en) 1985-01-30 1995-01-31 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter regulatory DNA sequence
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5817483A (en) 1985-03-30 1998-10-06 Stuart Kauffman Process for the production of stochastically-generated peptides,polypeptides or proteins having a predetermined property
EP0590689A2 (fr) 1985-03-30 1994-04-06 BALLIVET, Marc Procédé d'obtention d'ADN, ARN, peptides, polypeptides ou protéines, par une technique de recombinaison d'ADN
US5976862A (en) 1985-03-30 1999-11-02 Ixsys Corporation Process for obtaining DNA, RNA, peptides, polypeptides, or proteins, by recombinant DNA technique
US5824514A (en) 1985-03-30 1998-10-20 Stuart A. Kauffman Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides
EP0229046A1 (fr) 1985-03-30 1987-07-22 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn.
WO1986005803A1 (fr) 1985-03-30 1986-10-09 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn
US5814476A (en) 1985-03-30 1998-09-29 Stuart Kauffman Process for the production of stochastically-generated transcription or translation products
US5723323A (en) 1985-03-30 1998-03-03 Kauffman; Stuart Alan Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
US4668218A (en) 1985-04-12 1987-05-26 Aktiebolaget Draco Indicating means for a dosage dispensing device
US4766067A (en) 1985-05-31 1988-08-23 President And Fellows Of Harvard College Gene amplification
EP0218868A2 (fr) 1985-08-29 1987-04-22 New York Blood Center, Inc. Préparation de facteur nécrotique tumoral humain pur et hybridomes produisant des anticorps monoclonaux contre le facteur nécrotique tumoral humain
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5595898A (en) 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
US5693493A (en) 1985-11-01 1997-12-02 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5618920A (en) 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5698417A (en) 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5698435A (en) 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5122464A (en) 1986-01-23 1992-06-16 Celltech Limited, A British Company Method for dominant selection in eucaryotic cells
US5770359A (en) 1986-01-23 1998-06-23 Celltech Therapeutics Limited Recombinant DNA sequences, vectors containing them and method for the use thereof
US5827739A (en) 1986-01-23 1998-10-27 Celltech Therapeutics Limited Recombinant DNA sequences, vectors containing them and method for the use thereof
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
EP0237507A1 (fr) 1986-03-07 1987-09-16 Aktiebolaget Draco Dispositif d'inhalation de poudre
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US4767402A (en) 1986-07-08 1988-08-30 Massachusetts Institute Of Technology Ultrasound enhancement of transdermal drug delivery
US4925673A (en) 1986-08-18 1990-05-15 Clinical Technologies Associates, Inc. Delivery systems for pharmacological agents encapsulated with proteinoids
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5455030A (en) 1986-09-02 1995-10-03 Enzon Labs, Inc. Immunotheraphy using single chain polypeptide binding molecules
US5518889A (en) 1986-09-02 1996-05-21 Enzon Labs Inc. Immunoassay methods using single polypeptide chain binding molecules
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5534621A (en) 1986-09-02 1996-07-09 Enzon Labs, Inc. Immunoaffinity purification methods using single polypeptide chain binding molecules
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5231024A (en) 1986-09-13 1993-07-27 Basf Aktiengesellschaft Monoclonal antibodies against human tumor necrosis factor (tnf), and use thereof
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US4921794A (en) 1987-01-14 1990-05-01 President And Fellows Of Harvard College T7 DNA polymerase
US4795699A (en) 1987-01-14 1989-01-03 President And Fellows Of Harvard College T7 DNA polymerase
US5994616A (en) 1987-02-17 1999-11-30 Pharming B.V. Targeted synthesis of protein in mammary gland of a non-human transgenic mammal
US5304489A (en) 1987-02-17 1994-04-19 Genpharm International, Inc. DNA sequences to target proteins to the mammary gland for efficient secretion
US5565362A (en) 1987-02-17 1996-10-15 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
EP0288088A2 (fr) 1987-04-24 1988-10-26 Teijin Limited Détection du facteur nécrosant de tumeur; anticorps monoclonal et composition
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0550400A2 (fr) 1987-07-24 1993-07-07 Xoma Corporation Ensemble modulaire de gènes d'anticorps, anticorps ainsi préparés et leurs utilisations
EP0371998A1 (fr) 1987-07-24 1990-06-13 Int Genetic Eng Ensemble modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
US4939666A (en) 1987-09-02 1990-07-03 Genex Corporation Incremental macromolecule construction methods
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1989006283A1 (fr) 1988-01-11 1989-07-13 Ingene (International Genetic Engineering, Inc.) Nouveau vecteur de plasmide avec sequence a fonction de signal pour pectate lyase
US6010902A (en) 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
US4956288A (en) 1988-04-22 1990-09-11 Biogen, Inc. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA
US5770198A (en) 1988-05-18 1998-06-23 The Research Foundation Of The State Of New York Platelet-specific chimeric 7E3 immunoglobulin
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5601819A (en) 1988-08-11 1997-02-11 The General Hospital Corporation Bispecific antibodies for selective immune regulation and for selective immune cell binding
US5571698A (en) 1988-09-02 1996-11-05 Protein Engineering Corporation Directed evolution of novel binding proteins
US5837500A (en) 1988-09-02 1998-11-17 Dyax, Corp. Directed evolution of novel binding proteins
US5403484A (en) 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5142033A (en) 1988-09-23 1992-08-25 Hoffmann-La Roche Inc. Structure-independent DNA amplification by the polymerase chain reaction
US5066584A (en) 1988-09-23 1991-11-19 Cetus Corporation Methods for generating single stranded dna by the polymerase chain reaction
US5091310A (en) 1988-09-23 1992-02-25 Cetus Corporation Structure-independent dna amplification by the polymerase chain reaction
WO1990004036A1 (fr) 1988-10-12 1990-04-19 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
EP0438474B1 (fr) 1988-10-12 1996-05-15 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
WO1990003809A1 (fr) 1988-10-12 1990-04-19 Rochal Industries, Inc. Bandage conforme et matiere d'enduction
EP0368684A1 (fr) 1988-11-11 1990-05-16 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US4994370A (en) 1989-01-03 1991-02-19 The United States Of America As Represented By The Department Of Health And Human Services DNA amplification technique
US5116964A (en) 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5225538A (en) 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
US5266491A (en) 1989-03-14 1993-11-30 Mochida Pharmaceutical Co., Ltd. DNA fragment and expression plasmid containing the DNA fragment
US6060285A (en) 1989-03-23 2000-05-09 Roche Diagnostics Gmbh Process for the production of hetero-bispecific antibodies
WO1990014424A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Procede d'isolement de recepteurs presentant une specificite preselectionnee
WO1990014443A1 (fr) 1989-05-16 1990-11-29 Huse William D Coexpression de recepteurs heteromeres
WO1990014430A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique
WO1991000360A1 (fr) 1989-06-29 1991-01-10 Medarex, Inc. Reactifs bispecifiques pour le traitement du sida
WO1991002078A1 (fr) 1989-08-07 1991-02-21 Peptide Technology Ltd Ligands de liaison du facteur de necrose de tumeurs
US5643768A (en) 1989-10-05 1997-07-01 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5658754A (en) 1989-10-05 1997-08-19 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5770222A (en) 1989-12-22 1998-06-23 Imarx Pharmaceutical Corp. Therapeutic drug delivery systems
EP0463151B1 (fr) 1990-01-12 1996-06-12 Cell Genesys, Inc. Generation d'anticorps xenogeniques
EP0710719A1 (fr) 1990-01-12 1996-05-08 Cell Genesys, Inc. Génération d'anticorps xénogemiques
US5496549A (en) 1990-04-02 1996-03-05 Takeda Chemical Industries, Ltd. Bispecific monoclonal antibodies, thrombolytic agent and method of cell lysis
US5580717A (en) 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
WO1992000373A1 (fr) 1990-06-29 1992-01-09 Biosource Genetics Corporation Production de melanines a l'aide de microorganismes transformes
US5580734A (en) 1990-07-13 1996-12-03 Transkaryotic Therapies, Inc. Method of producing a physical map contigous DNA sequences
US5627052A (en) 1990-08-02 1997-05-06 B.R. Centre, Ltd. Methods for the production of proteins with a desired function
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
GB2272440A (en) 1990-08-29 1994-05-18 Genpharm Int Transgenic non-human animals capable of producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1992007076A1 (fr) 1990-10-18 1992-04-30 The Mathilda And Terence Kennedy Institute Of Rheumatology Recepteur de tnfalpha (facteur de necrose tumorale alpha) humain modifie
US5959084A (en) 1990-10-29 1999-09-28 Chiron Corporation Bispecific antibodies, methods of production and uses thereof
US6106833A (en) 1990-10-29 2000-08-22 Chiron Corporation Bispecific antibodies, methods of production and use thereof
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5582996A (en) 1990-12-04 1996-12-10 The Wistar Institute Of Anatomy & Biology Bifunctional antibodies and method of preparing same
US5404871A (en) 1991-03-05 1995-04-11 Aradigm Delivery of aerosol medications for inspiration
WO1992016221A1 (fr) 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
US5959083A (en) 1991-06-03 1999-09-28 Behringwerke Aktiengellschaft Tetravalent bispecific receptors, the preparation and use thereof
US5458135A (en) 1991-07-02 1995-10-17 Inhale Therapeutic Systems Method and device for delivering aerosolized medicaments
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
US5733761A (en) 1991-11-05 1998-03-31 Transkaryotic Therapies, Inc. Protein production and protein delivery
US5641670A (en) 1991-11-05 1997-06-24 Transkaryotic Therapies, Inc. Protein production and protein delivery
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5766886A (en) 1991-12-13 1998-06-16 Xoma Corporation Modified antibody variable domains
US5989530A (en) 1992-03-10 1999-11-23 Goldwell Ag Bleaching composition for human hair and process for its production
US5447851B1 (en) 1992-04-02 1999-07-06 Univ Texas System Board Of Dna encoding a chimeric polypeptide comprising the extracellular domain of tnf receptor fused to igg vectors and host cells
US5447851A (en) 1992-04-02 1995-09-05 Board Of Regents, The University Of Texas System DNA encoding a chimeric polypeptide comprising the extracellular domain of TNF receptor fused to IgG, vectors, and host cells
WO1994006498A1 (fr) 1992-09-23 1994-03-31 Fisons Plc Inhalateur
US6193967B1 (en) 1992-10-02 2001-02-27 Peter M. Morganelli Bispecific reagents for redirected targeting of human lipoproteins
WO1994008552A2 (fr) 1992-10-19 1994-04-28 Dura Pharmaceuticals, Inc. Inhalateur pour poudre seche
US5839446A (en) 1992-10-28 1998-11-24 Transmedica International, Inc. Laser perforator
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
US5849695A (en) 1993-01-13 1998-12-15 The Regents Of The University Of California Parathyroid hormone analogues useful for treatment of osteoporosis and disorders of calcium meatabolism in mammals
WO1994016970A1 (fr) 1993-01-19 1994-08-04 Glaxo Group Limited Dispositif
US6132992A (en) 1993-02-01 2000-10-17 Bristol-Myers Squibb Co. Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell
US5871753A (en) 1993-02-12 1999-02-16 Board Of Trustees Of The Leland Stanford Junior University Regulated transcription of targeted genes and other biological events
US5770428A (en) 1993-02-17 1998-06-23 Wisconsin Alumni Research Foundation Chimeric retrovial expression vectors and particles containing a simple retroviral long terminal repeat, BLV or HIV coding regions and cis-acting regulatory sequences, and an RNA translational enhancer with internal ribsome entry site
EP0614989A1 (fr) 1993-02-17 1994-09-14 MorphoSys AG Procédé de sélection in vivo des protéines attachées des ligands
WO1994025585A1 (fr) 1993-04-26 1994-11-10 Genpharm International, Inc. Animaux transgeniques capables de produire des anticorps heterologues
US5514670A (en) 1993-08-13 1996-05-07 Pharmos Corporation Submicron emulsions for delivery of peptides
US5625825A (en) 1993-10-21 1997-04-29 Lsi Logic Corporation Random number generating apparatus for an interface unit of a carrier sense with multiple access and collision detect (CSMA/CD) ethernet data network
US5643759A (en) 1993-10-30 1997-07-01 Biotest Pharma Gmbh Method for preparing bispecific monoclonal antibodies
WO1995016027A1 (fr) 1993-12-06 1995-06-15 Bioinvent International Ab Procede de selection de bacteriophages specifiques
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5849992A (en) 1993-12-20 1998-12-15 Genzyme Transgenics Corporation Transgenic production of antibodies in milk
US5833985A (en) 1994-03-07 1998-11-10 Medarex, Inc. Bispecific molecules for use in inducing antibody dependent effector cell-mediated cytotoxicity
WO1996007754A1 (fr) 1994-09-02 1996-03-14 The Scripps Research Institute Procedes de production de banques d'anticorps au moyen de chaines legeres d'immunoglobuline universelles ou rendues alleatoires
US5767260A (en) 1994-10-13 1998-06-16 Enzon Inc. Antigen-binding fusion proteins
US5763733A (en) 1994-10-13 1998-06-09 Enzon, Inc. Antigen-binding fusion proteins
US5821333A (en) 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
US5807706A (en) 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US6037453A (en) 1995-03-15 2000-03-14 Genentech, Inc. Immunoglobulin variants
US5656730A (en) 1995-04-07 1997-08-12 Enzon, Inc. Stabilized monomeric protein compositions
WO1996034096A1 (fr) 1995-04-28 1996-10-31 Abgenix, Inc. Anticorps humains derives de xeno-souris immunisees
US5814599A (en) 1995-08-04 1998-09-29 Massachusetts Insitiute Of Technology Transdermal delivery of encapsulated drugs
US5851198A (en) 1995-10-10 1998-12-22 Visionary Medical Products Corporation Gas pressured needle-less injection device and method
WO1997013852A1 (fr) 1995-10-10 1997-04-17 Genpharm International, Inc. Animaux non humains transgeniques pouvant produire des anticorps heterologues
WO1997022376A1 (fr) 1995-12-20 1997-06-26 Intersurgical Limited Nebuliseur
WO1997025086A2 (fr) 1996-01-03 1997-07-17 Glaxo Group Limited Dispositif d'inhalation
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
EP0814259A1 (fr) 1996-06-19 1997-12-29 Motorenfabrik Hatz GmbH & Co. KG Dispositif de démarrage à froid
US6210668B1 (en) 1996-09-03 2001-04-03 Gsf Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Destruction of contaminating tumor cells in stem cell transplants using bispecific antibodies
WO1998024884A1 (fr) 1996-12-02 1998-06-11 Genpharm International Animaux transgeniques non humains capables de produire des anticorps heterologues
WO1998024893A2 (fr) 1996-12-03 1998-06-11 Abgenix, Inc. MAMMIFERES TRANSGENIQUES POSSEDANT DES LOCI DE GENES D'IMMUNOGLOBULINE D'ORIGINE HUMAINE, DOTES DE REGIONS VH ET Vλ, ET ANTICORPS PRODUITS A PARTIR DE TELS MAMMIFERES
US5879681A (en) 1997-02-07 1999-03-09 Emisphere Technolgies Inc. Compounds and compositions for delivering active agents
WO1998035888A1 (fr) 1997-02-13 1998-08-20 Glaxo Group Limited Valve doseuse pour aerosols
WO1998050433A2 (fr) 1997-05-05 1998-11-12 Abgenix, Inc. Anticorps monoclonaux humains contre le recepteur du facteur de croissance epidermique
WO1998053847A1 (fr) 1997-05-29 1998-12-03 Ben Gurion University Of The Negev Research And Development Authority Systeme de transport transdermique
US7250165B2 (en) 2000-08-07 2007-07-31 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US9321836B2 (en) 2000-08-07 2016-04-26 Janssen Biotech, Inc. Anti-TNF antibodies, compositions, methods and uses
US7521206B2 (en) 2000-08-07 2009-04-21 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US7691378B2 (en) 2000-08-07 2010-04-06 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US7815909B2 (en) 2000-08-07 2010-10-19 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US7820169B2 (en) 2000-08-07 2010-10-26 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US8241899B2 (en) 2000-08-07 2012-08-14 Centocor, Inc. Anti-TNF antibodies, compositions, methods and uses
US8603778B2 (en) 2000-08-07 2013-12-10 Janssen Biotech, Inc. Anti-TNF antibodies, compositions, methods and uses
US9828424B2 (en) 2000-08-07 2017-11-28 Janssen Biotech, Inc. Anti-TNF antibodies, compositions, methods and uses
WO2002012500A2 (fr) 2000-08-07 2002-02-14 Centocor, Inc. Anticorps, compositions, procedes anti-il-12 et leur utilisation
US9102989B2 (en) 2002-05-23 2015-08-11 Cognis Ip Management Gmbh Non-revertible β-oxidation blocked Candida tropicalis
US9105939B2 (en) 2008-03-10 2015-08-11 Nissan Motor Co., Ltd. Battery with battery electrode and method of manufacturing same
US9107149B2 (en) 2010-04-07 2015-08-11 Lenovo (Singapore) Pte Ltd Wireless portable computer capable of autonomously adjusting load of wireless base station
US9109630B2 (en) 2013-01-15 2015-08-18 Jtekt Corporation Rolling bearing unit
US9401234B2 (en) 2013-03-22 2016-07-26 Polytronics Technology Corp. Over-current protection device
US9618978B2 (en) 2013-10-03 2017-04-11 Acer Incorporated Methods for controlling a touch panel and portable computers using the same
WO2017136524A2 (fr) * 2016-02-05 2017-08-10 Janssen Biotech, Inc. Anticorps anti-tnf, compositions, méthodes et utilisation pour le traitement ou la prévention du diabète de type 1
US9816280B1 (en) 2016-11-02 2017-11-14 Matthew Reitnauer Portable floor

Non-Patent Citations (106)

* Cited by examiner, † Cited by third party
Title
"Merck Manual", 1972, MERCK & COMPANY
"PDR Pharmacopoeia", 2000, TARASCON PUBLISHING, article "Tarascon Pocket Pharmacopoeia 2000"
"Pharmacotherapy Handbook", 1998, APPLETON AND LANGE
"Physician's Desk Reference", MEDICAL ECONOMICS, 1998
"Tarascon Pocket Pharmacopoeia 2000", 2000, TARASCON PUBLISHING, article "PDR Pharmacopoeia"
ASHKENAZI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 10535 - 10539
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1987, GREENE PUBLISHING ASSOC. AND WILEY-INTERSCIENCE
BABCOOK ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 7843 - 7848
BAKER ET AL., EUR. J. IMMUNOL., vol. 24, 1994, pages 2040 - 2048
BEBBINGTON ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 169 - 175
BERZOFSKY ET AL.: "Fundamental Immunology", 1984, RAVEN PRESS, article "Antibody-Antigen Interactions"
BOSHART ET AL., CELL, vol. 41, 1985, pages 521 - 530
BRINGMAN ET AL., HYBRIDOMA, vol. 6, 1987, pages 489 - 507
BUTLER ET AL., CYTOKINE, vol. 6, no. 6, 1994, pages 616 - 623
CAPELLAS ET AL., BIOTECHNOL. BIOENG., vol. 56, no. 4, 1997, pages 456 - 463
CAPON ET AL., NATURE, vol. 337, 1989, pages 525 - 531
CARTER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 4285
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901
CONRAD ET AL., PLANT MOL. BIOL., vol. 38, 1998, pages 101 - 109
CORCORAN ET AL., EUR. J. BIOCHEM., vol. 223, 1994, pages 831 - 840
CRAMER ET AL., CURR. TOP. MICROBOL. IMMUNOL., vol. 240, 1999, pages 95 - 118
CULLEN ET AL., MOLEC. CELL. BIOL., vol. 5, 1985, pages 438 - 447
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
E. GOLDBAUM: "Newly diagnosed children, young adults with Type 1 diabetes preserve endogenous insulin production with anti-TNF drug", 15 June 2020 (2020-06-15), XP002800776, Retrieved from the Internet <URL:http://www.buffalo.edu/news/releases/2020/06/023.html> [retrieved on 20201021] *
ELLIOTT ET AL., LANCET, vol. 344, 1994, pages 1125 - 1127
ENDOCRINE PRACTICE, vol. 24, no. 2, February 2018 (2018-02-01), pages 195 - 204
ENGELMANN, H. ET AL., J. BIOL. CHEM., vol. 265, 1990, pages 1531 - 1536
EREN ET AL., IMMUNOL., vol. 93, 1998, pages 154 - 161
F. W. ALT ET AL., J. BIOL. CHEM., vol. 253, 1978, pages 1357 - 1370
FISCH ET AL., BIOCONJUGATE CHEM., vol. 3, 1992, pages 147 - 153
FISCHER ET AL., BIOTECHNOL. APPL. BIOCHEM., vol. 30, October 1999 (1999-10-01), pages 99 - 108
FISHWALD ET AL., NATBIOTECHNOL, vol. 14, no. 7, 1996, pages 845 - 851
FISHWILD ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 826 - 851
GRAY ET AL., J. IMM. METH., vol. 182, 1995, pages 155 - 163
GREEN ET AL., NATURE GENETICS, vol. 7, 1994, pages 13 - 21
HANES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, May 1997 (1997-05-01), pages 4937 - 4942
HANES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, November 1998 (1998-11-01), pages 14130 - 14135
HARLOW ET AL.: "antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
HEROLD ET AL., N ENGL J MED., vol. 381, no. 7, 15 August 2019 (2019-08-15), pages 603 - 613
HIRAI ET AL., J. IMMUNOL. METH., vol. 96, 1987, pages 57 - 62
HOOD ET AL., ADV. EXP. MED. BIOL., vol. 464, 1999, pages 127 - 147
ITOH ET AL., BIOORG. CHEM., vol. 24, no. 1, 1996, pages 59 - 68
J. L. HAMLINC. MA, BIOCHEM. ET BIOPHYS. ACTA, vol. 1097, 1990, pages 107 - 143
JANSEN RESEARCH DEVELOPMENT: "A Study of SIMPONI to Arrest Beta-cell Loss in Type 1 Diabetes - Full Text View - ClinicalTrials.gov", 27 July 2016 (2016-07-27), XP055742582, Retrieved from the Internet <URL:https://clinicaltrials.gov/ct2/show/NCT02846545> [retrieved on 20201021] *
JONAK ET AL., PROGRESS BIOTECH, vol. 5
JONES ET AL., NATURE, vol. 321, 1986, pages 522
JUNGINGER ET AL.: "Drug Permeation Enhancement", 1994, MARCEL DEKKER, INC., pages: 59 - 90
KATSAROU ET AL.: "Type 1 diabetes mellitus", NATURE REVIEWS DISEASE PRIMERS, vol. 3, 2017
KATSUBE, Y. ET AL., INTJMOL. MED, vol. 1, no. 5, 1998, pages 863 - 868
KENNY ET AL., BIO/TECHNOL., vol. 13, 1995, pages 787 - 790
KNIGHT DM ET AL., MOLECULAR IMMUNOLOGY, vol. 30, 1993, pages 1443 - 1453
KOLLS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 215 - 219
KOZBOR ET AL., IMMUNOL. TODAY, vol. 4, 1983, pages 72 - 79
KUMARAN ET AL., PROTEIN SCI., vol. 6, no. 10, 1997, pages 2233 - 2241
L. MASTRANDREA ET AL: "Etanercept Treatment in Children With New-Onset Type 1 Diabetes: Pilot randomized, placebo-controlled, double-blind study", DIABETES CARE, vol. 32, no. 7, 1 July 2009 (2009-07-01), US, pages 1244 - 1249, XP055609403, ISSN: 0149-5992, DOI: 10.2337/dc09-0054 *
LACHIN ET AL., DIABETES, vol. 63, no. 2, 2014, pages 739 - 748
LACHIN ET AL., DIABETES, vol. 63, no. 2, February 2014 (2014-02-01), pages 739 - 748
LESSLAUER ET AL., EUR. J. IMMUNOL., vol. 21, 1991, pages 2883 - 2886
LIANG ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 137, 1986, pages 847 - 854
LIS ET AL., ARCH MED SCI., vol. 10, no. 6, 22 December 2014 (2014-12-22), pages 1175 - 1185
LOETSCHER ET AL., CELL, vol. 61, 1990, pages 351 - 359
LONBERG ET AL., INT REV LMMUNOL, vol. 13, no. 1, 1995, pages 65 - 93
LONBERG ET AL., NATURE, vol. 368, 1994, pages 856 - 859
M. GOSSENH. BUJARD, PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
M. J. PAGEM. A. SYDENHAM, BIOTECHNOLOGY, vol. 9, 1991, pages 64 - 68
MA ET AL., PLANT PHYSIOL., vol. 109, 1995, pages 341 - 6
MA ET AL., TRENDS BIOTECHNOL., vol. 13, 1995, pages 522 - 7
MARRACK ET AL., SCIENCE, vol. 248, 1990, pages 705 - 711
MARRE ET AL., FRONT CELL DEV BIOL., vol. 3, 2015, pages 67
MENDEZ ET AL., NATURE GENETICS, vol. 15, 1997, pages 146 - 156
MIRMIRA ET AL., CURR DIAB REP, vol. 16, 2016, pages 95
MIRMIRA ET AL., CURR DIAB REP., vol. 16, no. 10, October 2016 (2016-10-01), pages 95
MOLLER, A. ET AL., CYTOKINE, vol. 2, no. 3, 1990, pages 162 - 169
MORTENSEN ET AL., DIABETES CARE, vol. 32, 2009, pages 1384 - 90
MULLER, METH. ENZYMOL., vol. 92, 1983, pages 589 - 601
MURPHY ET AL., BIOCHEM. J., vol. 227, 1991, pages 277 - 279
NEUBERGER, M., NATURE BIOTECH., vol. 14, 1996, pages 826
NGUYEN ET AL., MICROBIOL. IMMUNOL., vol. 41, 1997, pages 901 - 907
PALMER ET AL., DIABETES, vol. 53, no. 1, 2004, pages 250 - 264
PALMER ET AL., DIABETES, vol. 53, no. 1, January 2004 (2004-01-01), pages 250 - 64
PEPPEL ET AL., J. EXP. MED., vol. 174, 1991, pages 1483 - 1489
PINCKNEY ET AL., CLIN THER., vol. 38, no. 6, 2016, pages 1327 - 1339
POWELL ET AL., BIOTECHNOL., vol. 8, 1990, pages 333 - 337
PRESTA ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323
SANDHU ET AL., CRIT. REV. BIOTECHNOL., vol. 16, 1996, pages 95 - 118
SCALLON ET AL., CYTOKINE, vol. 7, 1995, pages 759 - 770
SIMS ET AL., DIABETES CARE, vol. 39, 2016, pages 1519 - 1526
SMITH ET AL., J. MOL. BIOL., vol. 224, 1992, pages 899 - 904
SPRAGUE ET AL., J. VIROL., vol. 45, 1983, pages 773 - 781
STEENBAKKERS ET AL., MOLEC. BIOL. REPORTS, vol. 19, 1994, pages 125 - 134
STEFFES ET AL., DIABETES CARE, vol. 26, no. 3, 2003, pages 832 - 836
SURESH ET AL., METHODS IN ENZYMOLOGY, vol. 121, 1986, pages 210
TAYLOR ET AL., INT. IMMUNOL., vol. 6, no. 4, 1994, pages 579 - 591
TAYLOR ET AL., INTERNATIONAL IMMUNOLOGY, vol. 6, 1993, pages 579 - 591
TAYLOR ET AL., INTL. IMMUNOL., vol. 6, 1993, pages 579 - 591
TAYLOR ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, no. 23, 1992, pages 6287 - 6295
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655
TUAILLON ET AL., PROC NATL ACAD SCI USA, vol. 90, no. 8, 1993, pages 3720 - 3724
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534
VOS ET AL., SCIENCE, vol. 255, 1992, pages 306 - 312
WEN ET AL., J. IMMUNOL., vol. 17, 1987, pages 887 - 892
WERLEN ET AL., BIOCONJUGATE CHEM., vol. 5, 1994, pages 411 - 417
WHITELAM ET AL., BIOCHEM. SOC. TRANS., vol. 22, 1994, pages 940 - 944
WOOD ET AL., FEMS MICROBIOLOGY IMMUNOLOGY, vol. 76, 1991, pages 121 - 134
YPSOMED: "Go for convenience", 2 October 2018 (2018-10-02), XP055742584, Retrieved from the Internet <URL:https://yds.ypsomed.com/files/media/03_Documents/07_VarioJect/Ypsomed-VarioJect-Brochure.pdf> [retrieved on 20201021] *

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