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WO1996036883A1 - Dosage immunologique dans le plasma de la proteine similaire au glucagon, de type 1 (glp-1) - Google Patents

Dosage immunologique dans le plasma de la proteine similaire au glucagon, de type 1 (glp-1) Download PDF

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Publication number
WO1996036883A1
WO1996036883A1 PCT/DK1996/000212 DK9600212W WO9636883A1 WO 1996036883 A1 WO1996036883 A1 WO 1996036883A1 DK 9600212 W DK9600212 W DK 9600212W WO 9636883 A1 WO9636883 A1 WO 9636883A1
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WO
WIPO (PCT)
Prior art keywords
glp
antibody
directed against
terminal
mpgf
Prior art date
Application number
PCT/DK1996/000212
Other languages
English (en)
Inventor
Lone Pridal
Lennart Andersen
Flemming Stig Larsen
David Owens
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU56852/96A priority Critical patent/AU5685296A/en
Publication of WO1996036883A1 publication Critical patent/WO1996036883A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/605—Glucagons

Definitions

  • Immuno assay methods which specifically measures the amount of biological active GLP-1 .
  • the invention includes the following methods:
  • a sandwich immuno assay employing two different antibodies, directed against two different epitopes: one antibody directed against the C-terminai region and one antibody directed against the N-terminal region.
  • the C-terminal antibody must be specific i.e must cross-react less than 20% (preferably less than 5%) with both MPGF and GLP-K7-32) and the N-terminal antibody must cross-react less than 20% (preferably less than 5 %) with GLP-K9-37);
  • a sandwich immuno assay employing two different antibodies, directed against two different epitopes: one antibody directed against the C-terminal region and one antibody directed against the N-terminal region.
  • the N-terminal antibody must be specific i.e. must cross-react less than 20% (preferably less than 5%) with both MPGF and GLP-K9-37) and the C-terminal antibody must cross-react less than 20% (preferably less than 5 %) with GLP-K7-32),
  • MPGF and/or other fragments of MPGF lacking the biological activity of GLP-1 can be removed before assay by an immuno assay. This can be done in several ways.
  • MPGF can be removed by immunoabsorption with antibodies directed to an epitope in MPGF which is not present in biological active GLP-1 .
  • MPGF can be removed by chromatographic e.g. by fractionation of a plasma sample on HPLC.
  • the antibody combination in the sandwich immuno assay must be specific for biological active GLP-1 or the inactive GLP-1 immunoreac- tivity must be removed before assay.
  • GLP - 1 Glucagon - like peptide - 1 , GLP - 1 , is a recently discovered gut hor ⁇ mone [1 ,2].
  • Two biologically active forms of GLP - 1 are formed by post -translational processing of the precursor peptide pro - glucagon: GLP - 1 (7 - 36)amide and GLP - 1 (7 - 37), corresponding to proglucagon- (78 - 107)ar ⁇ ide and proglucagon(78 - 108), respectively [3]. Both peptides are present in plasma. However, in humans GLP - 1 (7 - 36)- amide is by far the most abundant form [3].
  • the amino acid sequence of GLP - 1 (7-37) is [4]:
  • GLP - 1 (7 - 36)amide is secreted from the the L-cells in the distal ileum as a response to oral intake of e.g. carbohydrates [1 ,2,5].
  • the peptide has several biological functions, with its main target in the islets of Langerhans in the endocrine pancreas. In ⁇ - cells, it enhances the glucose -stimulated insulin secretion [1 ,2] and the biosynthesis of insulin [6]. In D - cells, it enhances the secretion of somatostatin [1 ,2] and in a — cells, glucagon secretion is inhibited, either directly or through an intraislet pathway involving somatostatin [1 ,2]. In the gastro - intestinal tract it inhibits gastric emptying and secretion [7].
  • Pro-glucagon is expressed primarily in two tissues, the L-cells as described above and in the ⁇ -cells in the islets of Langerhans in the endocrine pancreas. In the ⁇ -cells pro-glucagon is processed differently than in the intestine, fragments containing GLP-1 formed in the ⁇ -cells are: proglucagon(72-158) and proglucagon(72-107). Neither of these two fragments have any known biological activity [1].
  • GLP- 1 the biologically active forms of GLP- 1 are GLP-K7-37), GLP- 1(7-36)amide, GLP-K7-35) and GLP-1- (7-34) [10]. If the N-terminal histidine in position 7 or the lysine in position 34 are removed, the biological activity decreases by at least three orders of magnitude [10].
  • GLP -1(7 -37) and GLP-1(7-36)amide have been shown to be degraded to GLP -1(9 -37) and GLP-1(9-36)amide, respectively, in vitro, when incubated in human plasma by the enzyme Dipeptidyl Peptidase IV (11,12). The half-life for this conversion was 19.9 ⁇ 6.6 and 20.4 ⁇ 1.4 minutes for GLP-1 (7-37) and GLP-1 (7-36)amide, respectively.
  • GLP- 1(7-36)amide was infused intravenously to give plasma concentrations of
  • Radioimmunoassay employing one C-terminal directed antibody
  • Radioimmunoassay employing one mid-region directed antibody
  • Sandwich ELISA employing two different antibodies, directed against two different epitopes: an antibody directed against the C-terminal region and an antibody directed against the N-terminal region.
  • GLP-1 (9-36)amide/(9-37) have severe cross-reactions with GLP-1 (9-36)amide/(9-37), which are found in plasma in up to 10 times the concentration of GLP- 1 (7-36)amide/(7-37).
  • the object of the present invention is to provide a method of quantifying GLP-1 in biological fluids.
  • the method can be used in search for variability of pharmacokinetic parameters. Determining this variability during the pre-clinical and clinical development of new drugs for regi ⁇ stration purposes is a requirement. It also contributes to an individual optimization of the treatment during clinical research and after introduc ⁇ tion into the market. This strategy would clearly result in an improve ⁇ ment of the benefit-risk ratio.
  • MPGF and inactive fragments of GLP-1 in plasma samples is a major cause of erroneous measurements of GLP-1 .
  • the "perfect" assay for biological active GLP-1 would have no cross- reaction (less than 20% (preferably less than 5%)) with MPGF, GLP-1 - (9-37)/(9-36)amide and GLP-K7-32).
  • either the antibody specificity or the pretreatment of the samples will provide a method of specifically quantifying biological active GLP-1 .
  • these methods will be superior to other known assays.
  • the problems with the present assays make them unfit for use in pharmacokinetic studies.
  • MPGF can be done in several ways e.g. by immunoabsorption, chromatographic.
  • the invention will now be described by way of examples thereof:
  • Figure 1 and 2 shows a clinical study where 7 patients with Non Insulin
  • NIDDM Dependent Diabetes Mellitus
  • Figure 1 shows the plasma concentrations measured by a radioimmuno ⁇ assay employing one C-terminal directed antibody [14].
  • the antibody used has been shown to cross-react (> 10%) with GLP-1 (1 -36)amide, GLP-1 (8-36)amide, GLP-1 (9-36)amide and less than ⁇ 1 % GLP-K7-37), GLP-K7-35), GLP-K7-34) and GLP-K7-33), and ⁇ ⁇ 0.1 % with Glucagon.
  • Figure 2 shows the plasma concentrations measured by a sandwich ELISA [19],the assay employs a monoclonal mouse antibody directed against the (26-33) region and a polyclonal rabbit antibody directed against the (7-14) region as catching and detecting antibody, respecti ⁇ vely.
  • This antibody combination used has been shown to cross-react 0 10%) with MPGF, GLP-1 (1 -36)amide, GLP-K7-35) and GLP-K7-34), only to a minor extent ( ⁇ 10%) with GLP-K7-33), less than ⁇ 1 % with GLP-1 (8-36)amide and GLP-1 (9-36)amide and ⁇ ⁇ 0.1 % with GLP-K7- 32), GLP-K7-31 ), GLP-1 (10-36)amide, GLP-K1 1 -36)amide and
  • MPGF and inactive fragments of GLP-1 in plasma samples is a major cause of erroneous measurements of GLP-1.
  • the assay according to the invention for biological active GLP-1 would have no cross-reaction (less than 20% (preferably less than 5%)) with MPGF, GLP-1 (9-37)/(9-36)amide and GLP-K7-32).
  • An sandwich immuno assay employing two different antibodies, directed against two different epitopes: one antibody directed against the C-terminal region and one antibody directed against the N-terminal region.
  • the C-terminal antibody must be specific i.e must cross-react less than 20% (preferably less than 5%) with both MPGF and GLP-K7-32) and the N-terminal antibody must cross-react less than 20% (preferably less than 5%) with GLP-K9-37).
  • An sandwich immuno assay employing two different antibodies, directed against two different epitopes: one antibody directed against the C-terminal region and one antibody directed against the N-terminal region.
  • the N-terminal antibody must be specific i.e. must cross-react less than 20% (preferably less than 5%) with both MPGF and GLP-K9-37) and the C-terminal antibody must cross-react less than 20% (preferably less than 5%) with GLP-K7-32).
  • MPGF and/or other biological inactive fragments of MPGF can be removed before assay by an immuno assay. This can be done in several ways.
  • MPGF can be removed by immunoabsorption with antibodies directed to an epitope in MPGF which is not present in biological active GLP-1 .
  • Subsequently measurement of the amount of biological active GLP-1 can be done by using a sandwich immuno assay method employing two different antibodies, directed against two different epitopes: an antibody directed against the C-terminal region and an antibody directed against the N-terminal region.
  • the N-terminal antibody must cross-react less than 20% (preferably less than 5%) with GLP-K9-37) and the C-terminal antibody must cross-react less than 20% (preferably less than 5%) with GLP-K7-32).
  • MPGF and/or other fragments of MPGF lacking the GLP-1 biologi- cal activity can be removed by chromatographic e.g. by fractionation of a plasma sample on HPLC. Subsequently mea ⁇ surement of the amount of biological active GLP-1 can be done by using an immuno assay employing at least one antibody directed against an epitope in GLP-K7-37) or GLP-1 (7-36)amide.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Endocrinology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
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  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne une technique de dosage immunologique avec formation d'un complexe du type sandwich, qui permet de mesurer spécifiquement la quantité de GLP-1 biologiquement active. L'invention concerne également un prétraitement des échantillons avant le dosage immunologique de la GLP-1.
PCT/DK1996/000212 1995-05-17 1996-05-14 Dosage immunologique dans le plasma de la proteine similaire au glucagon, de type 1 (glp-1) WO1996036883A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56852/96A AU5685296A (en) 1995-05-17 1996-05-14 Immunoassay for glucagon like protein 1 (glp-1) in plasma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK56295 1995-05-17
DK0562/95 1995-05-17

Publications (1)

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WO1996036883A1 true WO1996036883A1 (fr) 1996-11-21

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1750754A4 (fr) * 2004-03-31 2010-09-22 Centocor Ortho Biotech Inc Corps mimetiques glp-1 humains, compositions, procedes et utilisations
WO2012100267A1 (fr) * 2011-01-21 2012-07-26 Ir2Dx, Inc. Biomarqueurs pour détermination rapide de l'efficacité d'un médicament
CN104267194A (zh) * 2014-09-23 2015-01-07 上海市东方医院 人胰高血糖素样肽-1、抗体及其试剂盒
US11208477B2 (en) 2019-04-01 2021-12-28 Novo Nordisk A/S Antibodies and use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011457A1 (fr) * 1990-01-24 1991-08-08 Buckley Douglas I Analogues de glp-1 utiles dans le traitement du diabete

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011457A1 (fr) * 1990-01-24 1991-08-08 Buckley Douglas I Analogues de glp-1 utiles dans le traitement du diabete

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOMEDICAL RESEARCH, Volume 11, No. 2, 1990, H. TAKAHASHI et al., "Radioimmunoassay for Glucagon-Like Peptide-1 in Human Plasma Using N-Terminal and C-Terminal Directed Antibodies: a Physiologic Insulinotropic Role of GLP-1(7-36 Amide)", pages 99-108. *
DIGESTION, Volume 54, 1993, L.O. UTTENTHAL et al., "A Sensitive Enzyme-Linked Immunosorbent Assay for Glucagon-Like Peptide 1", pages 395-396. *
DIGESTION, Volume 54, 1993, M. GHIGLIONE, "Monoclonal Antibodies to Glucagon-Like Peptide 1", pages 396-397. *
EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Volume 22, 1992, R. EISSELE et al., "Glucagon-Like Peptide-1 Cell in the Gastrointestinal Tract and Pancreas of Rat, Pig and Man", pages 283-291. *
HISTOCHEMISTRY, Volume 86, 1987, TH. KAUTH et al., "Immunohistochemical Localization of Glucagon-Like Peptide 1* Use of Poly- and Monoclonal Antibodies", pages 509-515. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, No. 29, July 1994, J.J. HOLST et al., "Proglucagon Processing in Porcine and Human Pancreas", pages 18827-18833. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1750754A4 (fr) * 2004-03-31 2010-09-22 Centocor Ortho Biotech Inc Corps mimetiques glp-1 humains, compositions, procedes et utilisations
WO2012100267A1 (fr) * 2011-01-21 2012-07-26 Ir2Dx, Inc. Biomarqueurs pour détermination rapide de l'efficacité d'un médicament
CN104267194A (zh) * 2014-09-23 2015-01-07 上海市东方医院 人胰高血糖素样肽-1、抗体及其试剂盒
US11208477B2 (en) 2019-04-01 2021-12-28 Novo Nordisk A/S Antibodies and use thereof

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