WO2014205617A1 - Peptide marqué au lanthanide et utilisation de celui-ci - Google Patents

Peptide marqué au lanthanide et utilisation de celui-ci Download PDF

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Publication number
WO2014205617A1
WO2014205617A1 PCT/CN2013/077729 CN2013077729W WO2014205617A1 WO 2014205617 A1 WO2014205617 A1 WO 2014205617A1 CN 2013077729 W CN2013077729 W CN 2013077729W WO 2014205617 A1 WO2014205617 A1 WO 2014205617A1
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group
gamma
absent
lanthanide
peg
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PCT/CN2013/077729
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English (en)
Inventor
Chun SONG
Jie Han
Chengcheng Zhang
Xin Zhao
Huizhen GENG
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Shandong University
Jinan Chengcheng Biotechnology Co., Ltd.
Suzhou Baikang Biotechnology Co., Ltd.
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Application filed by Shandong University, Jinan Chengcheng Biotechnology Co., Ltd., Suzhou Baikang Biotechnology Co., Ltd. filed Critical Shandong University
Priority to PCT/CN2013/077729 priority Critical patent/WO2014205617A1/fr
Publication of WO2014205617A1 publication Critical patent/WO2014205617A1/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • the present invention relates to lanthanide labeled peptide and polypeptide, such as lanthanide labeled insulin, insulin- like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide-1 (GLP-1), their derivatives and/or analogs, as well as their use in establishing robust biological assays.
  • lanthanide labeled peptide and polypeptide such as lanthanide labeled insulin, insulin- like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide-1 (GLP-1), their derivatives and/or analogs, as well as their use in establishing robust biological assays.
  • Lanthanide elements are 15 elements from lanthanum (La) to lutetium (Lu) corresponding to the progressive filling of the 4f electrons in a subshell.
  • Fluorescent lanthanide chelates can be divided into three main groups according to their luminescent characteristics that are based on the 4f energy levels. In the strong luminescence group (Sm 3+ , Eu 3+ , Tb 3+ , Dy 3+ chelates), there is a large energy difference between the excited level and the ground level of these metal ions. Non-radioactive transition does not occur easily so the luminescence quantum yield is high.
  • the energy difference between the excited and group levels of the metal ion is fairly small. Significant portion of the excitation energy is dissipated via non-radioactive thermal deactivation processes and the quantum yield is low.
  • the chelates of the remaining lanthanide elements, La 3+ , Gd 3+ , Lu 3+ are not luminescent.
  • lanthanide chelates that make them especially suited for time-resolved fluorescence assays are as follows: (1) Decay time of lanthanide chelates can exceed 1,000 000 ns. Fluorescence from lanthanide chelates may last up to 200,000 times longer than from conventional fluorophores. Non-specific background fluorescence from plates, cells, and reagents in many fluorescence measurements has a decay time of only about 10 ns. It thus dies away before the sample fluorescence is measured. In a time-resolved fluorometer or multilabel reader, the sample is pulsed 1000 per second with an excitation light of 340 nm.
  • the present invention provides various lanthanide labeled peptide and polypeptide, such as lanthanide labeled insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glucagon, glucagon-like peptide- 1 (GLP-1), their derivatives and/or analogs, as well as their use in establishing robust biological assays.
  • IGF-1 insulin-like growth factor 1
  • IGF-2 insulin-like growth factor 2
  • GLP-1 glucagon-like peptide- 1
  • amino acid encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylate groups are attached to the same carbon (the alpha carbon).
  • the alpha carbon optionally may have one or two further organic substituents.
  • designation of an amino acid without specifying its stereochemistry is intended to encompass either the L or D form of the amino acid, or a racemic mixture.
  • an amino acid is designated by its one letter code (i.e., K)
  • such a designation is intended to specify the native L form of the amino acid, whereas the D form will be specified by inclusion of a lower case d before the one letter code (i.e., dK).
  • substitution refers to the replacement of one amino acid residue by a different amino acid residue.
  • “Native insulin” means mammalian insulin (e.g., human insulin, bovine insulin, porcine insulin or whale insulin) from natural, synthetic, or genetically engineered sources.
  • Human insulin comprises a 21 amino acid A chain and a 30 amino acid B chain, which are cross-linked by disulfide bonds.
  • a properly cross-linked human insulin includes three disulfide bridges: one between A7 and B7, a second between A20 and B 19, and a third between A6 and Al l .
  • Insulin derivative refers to a modified insulin peptide, comprising an A chain and B chain dimer, as well as single-chain insulin analogs thereof, that retains close sequence homology with native insulin. "Insulin derivative” exhibits some, all or enhanced activity relative to a corresponding native insulin or is converted in vivo or in vitro into a polypeptide exhibiting some, all or enhanced activity relative to a corresponding native insulin. "Insulin derivative” differs by substitution and/or deletion of at least one naturally occurring amino acid residue and/or addition of at least one amino acid residue and/or organic residue from the corresponding, otherwise identical naturally occurring insulin.
  • insulin derivative Gly(A21), Arg(B31), Arg(B32) human insulin (insulin Glargine by Sanofi-Aventis).
  • Another example of an insulin derivative is insulin aspart, in which Pro(B28) is substituted with an aspartic acid residue.
  • insulin lispro Another example of an insulin derivative is insulin lispro, in which Pro(B28) and Lys(B29) on the C-terminal end of the B chain are reversed.
  • Insulin glulisine is a rapid-acting insulin analogue that differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid.
  • insulin derivatives are non-mammalian insulins.
  • insulin derivatives are known in the art. Unless the context specifically indicates otherwise (e.g., where a specific insulin is referenced, such as "human insulin” or the like), the term “insulin derivative” is used broadly to include native insulins and insulin derivatives.
  • Single chain insulin derivative encompasses a group of structurally-related proteins wherein the insulin A and B chains are covalently joined by a linker.
  • IGF-1 derivative comprises a peptide with a sequence as set forth in SEQ ID NO: 3 as well as derivatives thereof having 1-5 amino acid substitution, addition or deletion, with the proviso that the A chain and B chain do not each have the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • IGF-2 derivative comprises a peptide sequence of SEQ ID NO: 4 as well as derivatives thereof having 1-5 amino acid substitution, addition or deletion, with the proviso that the A chain and B chain do not each have the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • GLP-1 includes native GLP-1, a derivative or fragment of native GLP-1 peptide.
  • a GLP-1 derivative has the amino acid sequence of GLP-l(7-37)-OH or GLP-1 (7-36)-NH 2 or a fragment thereof modified so that 1,2,3,4,5 or 6 amino acids differ from the amino acid in the corresponding position of GLP-1 (7-37)-OH or GLP-l(7-36)-NH 2 .
  • GLP-1 derivative encompasses polypeptides having from about twenty-five to about thirty-nine naturally occurring or non-naturally occurring amino acids that have sufficient homology to native GLP-1 (7-37)-OH such that they exhibit insulinotropic activity by binding to the GLP-1 receptor on ⁇ -cells in the pancreas.
  • a GLP-1 derivative typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37)-OH, an analog of GLP-1 (7-37)-OH, a fragment of GLP-l(7-37)-OH or a fragment of a GLP-1 (7-37)-OH analog.
  • GLP-1 (7-37)-OH has the amino acid sequence of SEQ ID NO: 6
  • amino terminus of GLP-l(7-37)-OH has been assigned number 7 and the carboxy-terminus number 37.
  • GLP-1 derivatives also encompass "GLP-1 fragments.”
  • a GLP-1 fragment is a polypeptide obtained after truncation of one or more amino acids from the N-terminus and/or C-terminus of GLP-1 (7-37)-OH or an analog or derivative thereof.
  • the nomenclature used to describe GLP-1 (7-37)-OH is also applicable to GLP-1 fragments.
  • GLP-1 (9-36)-OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C-terminus. The amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-l(7-37)-OH.
  • the N-terminal glutamic acid in GLP-1 (9-36)-OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP-l(7-37)-OH.
  • GLP-1 (7-36)-OH the glycine at position 37 of GLP-l(7-37)-OH is deleted.
  • GLP-1 derivatives also include polypeptides in which one or more amino acids have been added to the N-terminus and/or C-terminus of GLP-l(7-37)-OH, or fragments or analogs thereof. It is preferred that GLP-1 derivatives of this type have up to about thirty-nine amino acids.
  • the amino acids in the "extended" GLP-1 derivative are denoted by the same number as the corresponding amino acid in GLP-1 (7-37)-OH.
  • the N-terminus amino acid of a GLP-1 derivative obtained by adding two amino acids to the N-terminal of GLP-1 (7-37)-OH is at position 5; and the C-terminus amino acid of a GLP-1 derivative obtained by adding one amino acid to the C-terminus of GLP-1 (7-37)-OH is at position 38.
  • position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 derivatives, as in GLP-l(7-37)-OH.
  • Amino acids 1-6 of an extended GLP-1 derivative are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-l(l-37)-OH.
  • Amino acids 38-45 of an extended GLP-1 derivative are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or Exendin-4.
  • GLP-1 derivatives are also defined as a molecule having the amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group.
  • a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine ⁇ -amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
  • Modifications of the terminal amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications.
  • Lower alkyl is C1-C4 alkyl.
  • one or more side groups, or terminal groups may be protected by protective groups known to the ordinarily-skilled protein chemist.
  • the ⁇ -carbon of an amino acid may be mono- or dimethylated.
  • native glucagon refers to native human glucagon having the sequence H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala -Gln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr-OH (SEQ ID NO: 5). Amino acids within the sequence can be considered to be numbered consecutively from 1 to 29 in the conventional N-terminal to C-terminal direction.
  • a "glucagon derivative” as used herein includes any peptide comprising, either the amino acid sequence as set forth in SEQ ID NO: 5, or any derivative thereof, including amino acid substitutions, additions, or deletions, or post translational modifications (e.g. methylation, acylation, ubiquitination and the like) of the peptide, that stimulates glucagon or GLP-I receptor activity.
  • Polyethylene glycol or “PEG”, refers to condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH 2 CH 2 ) n OH, wherein n is at least 1.
  • the present invention provides a lanthanide labeled (polypeptide, which comprises at least one lanthanide label and wherein said lanthanide is attached to the N terminal or Lysine side chain amino group of said (polypeptide.
  • said lanthanide label may comprise Sm 3+ , Eu 3+ , Tb 3+ , Dy 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Pm 3+ , Er 3+ , Tm 3+ or Yb 3+ chelate.
  • the present invention relates to an insulin derivative having an A chain comprising the sequence of GIVEQCCX 8 SICSLYQLENYCX 2 iX 22 (SEQ ID NO: 7) and a B chain comprising the sequence of
  • X 8 is selected from the group consisting of threonine and histidine; X 21 is asparagine, glycine or alanine; X 22 is absent or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • Ji is a lanthanide label or absent
  • Ri is an optional spacer between Ji and N terminal amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • X23-2 6 is phenylalanine-valine-asparagine-glutamine(SEQ ID NO: 49),
  • valine-asparagine-glutamine asparagine-glutamine, glutamine or absent;
  • X32 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • X 48 is tyrosine or absent, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • threonine or absent, or has the general structure
  • N is 1 ,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • X 5 o is selected from the group consisting of proline, lysine, or absent, or has the general structure
  • N is 1 ,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label
  • threonine, alanine or absent r has the general structure
  • N is 1 ,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • N is 1 ,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid; J is a lanthanide label, and
  • an insulin analog is provided, wherein the A chain of the insulin peptide comprises the sequence
  • GIVEQCCTSICSLYQLENYCN SEQ ID NO: 9 and the B chain comprises a sequence selected from the group consisting of: HLCGSHLVEALYLVCGERGFF (SEQ ID NO: 10), FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 11) and FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 12).
  • a lanthanide label is attached to either insulin B chain N terminal or B29 Lysine side chain amino group.
  • Our insulin receptor binding assay and insulin receptor phosphorylation assay results showed that lanthanide labeled insulin maintained full potency as native insulin.
  • single-chain insulin analogs are provided.
  • the carboxy terminus of the human insulin B chain, or a functional analog thereof is covalently linked to the N-terminus of an A chain analog of the present application.
  • the B chain is linked to the A chain via a peptide linker of 4-12 or 4-8 amino acids.
  • Said peptide linker is selected from the group consisting of:
  • Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 13), Gly-Tyr-Gly-Ser-Ser-Arg-Arg- Ala-Pro-Gln-Thr (SEQ ID NO: 14), Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO: 15), Gly-Gly-Gly-Gly-Gly-Lys-Arg (SEQ ID NO: 16), Arg-Arg-Gly-Gly-Gly-Gly- Gly (SEQ ID NO: 17), Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 18), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 19), Gly-Gly-Tyr-Pro-Gly- Asp-Val-Lys-Arg (SEQ ID NO: 20), Arg-Arg-Tyr-Pro
  • the present invention relates to an IGF-1 derivative having the following sequence:
  • Ri is an optional spacer between Ji and N terminal amino group. It is selected from the group consisting of long chain fatty acids, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ji is a lanthanide label or absent
  • X 2 7 is lysine, arginine, homoarginin r has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label
  • N is 1 ,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label
  • N is 1 ,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label
  • N is 1 ,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • J is a lanthanide label
  • a lanthanide label is attached to either IGF-1 B domain N terminal or Lysine (at positions 27, 65, 68) side chain amino group.
  • IGF-1 receptor binding assay and IGF-1 receptor phosphorylation assay results showed that lanthanide labeled IGF-1 maintained full potency as native IGF-1.
  • the present invention also relates to an IGF-2 derivative having the following sequence:
  • Ri is an optional spacer between Ji and N terminal amino group, and it is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ji is a lanthanide label or absent
  • X 30 is lysine, arginine, homoarginine or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • J is a lanthanide label or absent
  • X 65 is lysine, arginine, homoarginine, absent, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • X 68 is absent, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • J is a lanthanide label or absent
  • lanthanide label comprised in said IGF-2 derivative.
  • a lanthanide label is attached to either IGF-2 B domain N terminal or Lysine (at positions 30 and 65) side chain amino group.
  • the present invention relates to a glucagon derivative having the following sequence:
  • X 12 is lysine or arginine ;
  • X 21 is aspartic acid, lysine, cysteine, homocysteine, ornithine, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is selected from the group consisting of long chain fatty acid and polyethylene glycol (PEG);
  • J is a lanthanide label or absent
  • glutamine glutamine, lysine, cysteine, homocysteine, ornithine, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, where Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is selected from the group consisting of long chain fatty acid and polyethylene glycol (PEG);
  • J is a lanthanide label or absent
  • threonine lysine, cysteine, homocysteine, ornithine, or absent, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limieted to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide or vinylsulfone group.
  • Li is an optional spacer or linker. It is selected from long chain fatty acids or polyethylene glycol (PEG);
  • J is a lanthanide label or absent
  • lysine, cysteine, homocysteine or a short peptide sequence of 1 to 5 amino acid long, and one amino acid of which is lysine, cysteine, homocysteine, or absent, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);
  • J is a lanthanide label or absent
  • One embodiment of the present invention is directed to a glucagon derivative that has been modified relative to the native glucagon to improve the peptide solubility and stability in aqueous solutions, while retaining the native peptide's biological activity.
  • a glucagon derivative wherein the native glucagon sequence has been modified to contain a naturally occurring or synthetic amino acid in at least one of positions 16, 17, 20, 21 , 24 and 29 of the native sequence that is different from the corresponding amino acid of the native sequence.
  • one or more amino acids at position 16, 17, 20, 21 , 24 and 29 of the native sequence are substituted with an amino acid selected from the group consisting of lysine, arginine, cysteine, and ornithine.
  • the lysine residue at position 12 of the native peptide is substituted with arginine and an amino acid present at one of the positions 16, 17, 20, 21 , 24 and 29 is substituted by a single lysine.
  • the amino acid present at position 16, 17, 20, 21, 24 or 29 of the native peptide is substituted with cysteine.
  • positions of the native glucagon can be modified while retaining at least some of the activity of the parent peptide. Accordingly, one or more of the amino acids located at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide as set forth in SEQ ID NO: 5 can be substituted with an amino acid different from that present in the corresponding position of the native glucagon, while still retaining the biological activity of the native glucagon. In one embodiment, the substitutions at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27, 28 or 29 are conservative amino acid substitutions.
  • an amino acid substitution using a natural or synthetic amino acid is made at position 16, 17, 20, 21, 24 or 29 of the glucagon, wherein the substitute amino acid allows for the covalent attachment of a lanthanide label to the amino acid side chain.
  • a lanthanide label is bound to an amino acid side chain at position 16, 21 or 24 of the glucagon.
  • the substitution is made at position 21 or 24 of the glucagon.
  • a glucagon derivative that comprises a lanthanide label covalently bound to the side chain of an amino acid present at position 16, 17, 20, 21, 24 or 29, wherein the glucagon derivative further comprises one, two or three amino acid substitutions at positions selected from positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29.
  • the methionine residue present at position 27 of the native glucagon peptide is changed to leucine or norleucine to prevent oxidative degradation of the peptide.
  • the present invention also encompasses glucagon fusion peptides, wherein a second peptide has been fused to the c-terminus of the glucagon peptide.
  • the glucagon fusion peptide may comprise an amino acid sequence of SEQ ID NO: 45(GPSSGAPPPS), SEQ ID NO: 46(KRNRNNIA) or SEQ ID NO: 47(KRNR) linked to amino acid 29 of the glucagon peptide through a peptide bond.
  • the present invention also relates to a GLP-1 derivative having the following sequence:
  • X 8 is selected from the group consisting of alanine, 2-methylalanine (Aib), serine and glycine;
  • X 2 6 is lysine, arginine, cysteine, homoc steine, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);
  • J is lanthanide label or absent
  • rginine lysine, cysteine, homoc steine, or has the general structure
  • N is 1,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);
  • J is lanthanide label or absent
  • N is 1,2,3,4,5,6,7 or 8;
  • RL IS an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);
  • J is lanthanide label or absent
  • N 1,2,3,4,5,6,7 or 8;
  • R L is an optional spacer between J and the amino group. It is selected from the group consisting of long chain fatty acid, polyethylene glycol (PEG), beta-alanine, gamma-aminobutyric acid and gamma-glutamic acid;
  • PEG polyethylene glycol
  • beta-alanine beta-alanine
  • gamma-aminobutyric acid gamma-glutamic acid
  • Ri has a formula of Mi-Li-NH-, wherein Mi is a thiol reactive functional group, including but not limited to maleimide, haloacetyl (e.g. iodoacetyl), pyridyl disulfide and vinylsulfone group.
  • Li is an optional spacer or linker. It is long chain fatty acids or polyethylene glycol (PEG);
  • J is lanthanide label or absent
  • a lanthanide label generally consists of a chelator, a lanthanide metal ion, and a linking group connecting the label with a biomolecule.
  • a synergic agent such as tri-n-octylphosphine oxide (TOPO) or 1,10-phenanthroline, intensifies the emission through the formation of a ternary ⁇ -diketonate-Eu -TOPO complex.
  • TOPO tri-n-octylphosphine oxide
  • 1,10-phenanthroline intensifies the emission through the formation of a ternary ⁇ -diketonate-Eu -TOPO complex.
  • Another class of chelators consists of derivatives of pyridine, 2,2'-bipyridine, 2,2'2"-terpyridine, and 1,10-phenanthroline.
  • the chelators used in this invention also include, but not limited to, diethylenetriaminetetraacetic acid (DTTA), diethylenetriamine pentaacetic acid (DTPA), triethylenetetraamine hexaacetic acid (TTHA),
  • DTTA diethylenetriaminetetraacetic acid
  • DTPA diethylenetriamine pentaacetic acid
  • TTHA triethylenetetraamine hexaacetic acid
  • the lanthanide ions include Sm 3+ , Eu 3+ , Tb 3+ , Dy 3+ , Tm 3+ , Nd 3+ , Ho 3+ , Er 3+ , Yb 3+ , Pm 3+ , Pr 3+ , Ce 3+ .
  • linking group able to bind biomolecules.
  • Isothiocyanate, sulfonyl chloride, carboxylate of N-hydroxysuccinimide, and maleimide are usually used to couple with peptides, proteins and biological molecules.
  • These linking groups can be bound to an amino group or a thiol group of the biomolecules. Examples include
  • the DTTA group (diethylenetriamine tetraacetic acid) forms a stable complex with a lanthanide ion. Another example is
  • Bifunctional bridging reagents are also available for the conjugation of amino or thiol derivatives of lanthanide complexes with biomolecules.
  • the chelate chemistry used for lanthanides can be compatible with chelation of radiolabeled metals (e.g. m In, 99m Tc, 68 Ga) in lieu of photoactive lanthanides, allowing such agents to be used for ⁇ -ray or positron emission imaging in the field of nuclear medicine.
  • radiolabeled metals e.g. m In, 99m Tc, 68 Ga
  • the location and concentration of these radionuclide-labeled agents can be determined using positron emission tomography (PET) or single photon emission computed tomography (SPECT) scanning.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • Figure 1 schematically illustrates combination of insulin chains.
  • Figure 2 schematically illustrates lanthanide labeled insulin.
  • Figure 3 schematicall illustrates lanthanide labeled IGF-1.
  • Figure 4 illustrates the chemical structure of selected examples of lanthanide chelators.
  • Figure 5 shows that lanthanide labeled insulins remains active and their biological activity is comparable to that of the native insulin.
  • a chelator When a chelator is coupled to the side chain amino group of lysine residue on the solid phase, an orthogonal deprotection scheme was used. Lysine side chain amino group is preferably protected by allyloxycarbonyl (aloe) group. Upon completion of syntheisis of the peptide sequence, aloe group(s) is preferably removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2: 1 mixture of methylene chloride, acetic acid, and N-Methylmorpholine (NMM) for 2 hours.
  • aloe group(s) preferably removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2: 1 mixture of methylene chloride, acetic acid, and N-Methylmorpholine (NMM) for 2 hours.
  • an oxidative sulfitolysis step was employed, which involves addition of -SO 3 groups to the reduced sulfur residues on cysteines of peptides, preventing the formation of potentially incorrect disulfide bonds during the solubilization and early purification steps prior to correct refolding of the proteins under optimal conditions for renaturation.
  • Incorrect disulfide bond formation during the solubilization and renaturation processes of peptide production accounts for a significant decrease in yield.
  • G10 or G25 column was used for desalting.
  • a buffer was 0.05 M ammonium bicarbonate and B buffer was 0.05 M ammonium bicarbonate with 50% acetonitrile. The correct fractions were combined, frozen, and lyophilized. Combination of human insulin A and B chain s-sulfonates
  • a chain s-sulfonates and B chain s-sulfonates (2: 1, w/w) were dissolved in 0.1M glycine buffer (pH 10.5) at a peptide concentration of 5-10 mg/ml.
  • 1.2 molar equivalent (SH:SSC>3 ⁇ ) of dithiothreitol (DTT) was added. The reaction was stirred at
  • the following side chain protecting groups were used: Arg(Tos), Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl-Z), Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br-Z).
  • the completed peptidyl resin was treated with 20% piperidine/dimethylformamide to remove the Trp formyl protection and then transferred to an HF reaction vessel and dried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added along with a magnetic stir bar.
  • the vessel was attached to the HF apparatus (Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, and approx. 10 ml liquid hydrogen fluoride was condensed in.
  • the reaction unit was stirred in an ice bath for 1 hr, then the HF was removed in vacuo. The residue was suspended in ethyl ether; the solids were filtered, washed with ether, and the peptide was extracted into 50 ml aqueous acetic acid.
  • the peptide ligand was dissolved in water and the pH was adjusted to 5-6.
  • the ligand concentration was determined by UV absorptions at 280nM.
  • An equimolar amount of metal salt was added as aqueous solution and the pH was maintained at 5-6. After 30 minutes of stirring at room temperature, the pH was raised to 8 with ammonium bicarbonate solution.
  • the conjugate was purified on Sephadex G25 or alternatively RP-HPLC.
  • the labeled peptide was eluted from the column in acetonitrile gradient in 0.02-0.1 mol/L triethylammonium acetate (pH 7.5). Correct fractions were combined, frozen, and lyophilized.
  • Tris-based buffer Preferably, a Tris-based buffer is used.
  • HEPES and phosphate buffers may also be used.
  • the buffer contains preferably a blocking agent such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • purified BSA is preferred, or alternatively high grade of casein or ovalbumin may be used to block non-specific binding.
  • a detergent such as Tween 20 or Tween 40 is also needed in the buffer to further prevent non-specific binding to the plate.
  • the assay buffer should contain low concentrations of chelator such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). It is, however, essential to note that too much chelator present in the assay buffer will eventually start competing for the lanthanide and will render the assay unsuccessful. Preferably, no more than 50 ⁇ /L of chelator may be used.
  • chelator such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • an assay buffer composition for an assay was 50mM Tris-HCl, pH 7.5-8, containing 0.9% NaCl, 0.2-0.5% of purified BSA, 0.01-0.1% Tween (20 or 40) and 20 ⁇ EDTA.
  • Labeled peptides were stored at a high concentration and in the absence of chelators or competing metals in the buffer. Preferably, diluted reagents shall not be stored. In most cases, 50 mmol/L Tris-HCl buffered saline solution containing 0.1-0.5% purified BSA will ensure the stability of the labeled compound during storage. For storage purposes, phosphate buffers must not be used due to their chelating nature. Storage should be at the optimal temperature for the peptide. If the labeled peptide requires storage at +4°C, it is preferred to add a bacteriostatic agent such as sodium azide (NaN 3 ) at a concentration of 0.05-0.1%.
  • a bacteriostatic agent such as sodium azide (NaN 3 ) at a concentration of 0.05-0.1%.
  • Cells of Chinese hamster ovary (CHO) cell line were transfected with human insulin proreceptor gene coupled to a gene for methotrexate resistance.
  • Cells were cultured in Eagles' minimum essential medium (without deoxynucleosides) + 10% fetal bovine serum supplemented with G418 (400 ⁇ g/ml) and methotrexate (50 nM). Under these conditions cells expressed >10 7 IR molecules per cell.
  • the beads were transferred to a column and washed 2 with 50 mM HEPES, 0.5 M NaCl, 0.5% Octyl-P -D-glucopyranoside, pH 7.8, 1 mM PMSF, 2 ⁇ g/ml aprotinin, and the adsorbed glycoproteins were eluted with steps of 0.1 M, 0.2 M, 0.3 M N-acetyl glucosamine in the above buffer.
  • the peak fractions eluted with 0.3 M N-acetyl glucosamine were pooled, their protein contents were measured using Bradford or BCA assays (Bio-Rad, Richmond, CA), and their purity was assessed by SDS-PAGE.
  • the identity of the band at 94 kDa was confirmed by probing as Western blot with anti-IR ⁇ -chain antibody.
  • a typical preparation yielded 150-200 ⁇ g of IR.
  • Truncated soluble receptors Assays were performed by incubating the receptors in a total volume of 200 ⁇ ⁇ with
  • 125 I-IGF-l (3-10 pM) or 125 I-insulin (3 pM) and increasing concentrations of unlabelled ligand in binding buffer A [100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100] for 48 h at 4 °C. Subsequently, bound ligand was precipitated with 0.2 % gama-globulin and 500 ⁇ of 25 % (w/v) poly( ethylene glycol) 8000, and the radioactivity in the pellet was measured. The concentration of the receptors was adjusted to yield 15-20 % binding when no competing ligand was added in the competition assay.
  • the affinity of peptides to glucagon receptor was measured in a competition binding assay utilizing scintillation proximity assay technology.
  • Serial 3 -fold dilutions of the peptides made in scintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clear bottom plate (Corning Inc., Acton, Mass.) with 0.05 nM (3- [ 125 I]-iodotyrosyl) TyrlO glucagon (Amersham Biosciences, Piscataway, N.J.), 1-6 micrograms per well, plasma membrane fragments prepared from cells over-expressing human glucagon receptor, and 1 mg/well polyethyleneimine-treated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, N.J.).
  • GLP-1 derivatives The binding affinity of GLP-1 derivatives was determined in a setting of cellular ELISA. Briefly, INS-1 cells cultured in 96 well plates (BD Biosciences) at roughly 95% confluence were rinsed with PBS and fixed with 4% paraformaldehyde (Thermo Scientific) for 10 min at room temperature and quenched for 5 min with 2% glycine in PBS, pH 7.5. For the binding capacity experiment, cells were incubated with logarithmic dilutions of GLP-1 derivative alone (1 *10 ⁇ 5 to 1 * 10 ⁇ 12 M) for total binding or in combination with 10 ⁇ GLP-1 (Abeam Inc, USA) for non-specific binding.
  • GLP-1 derivative to bind to the GLPl receptor was assessed by a radioligand competition-binding assay with intact receptor-expressing human GLP-1 receptor-bearing CHO cells.
  • human GLP-1 receptor-bearing CHO cells (-200,000) were incubated for 1 hr at room temperature with a constant amount of
  • radioligand I-GLP1 (5 pM, -20,000 cpm) in the presence of increasing concentrations (ranging from 0 to 1 ⁇ ) of GLP-1 derivative in Krebs-Ringers/HEPES (KRH) medium (25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KC1, 2 mM CaCl 2 , 1 mM KH 2 P0 4 , 1.2 mM MgS0 4 ) containing 0.01% soybean trypsin inhibitor and 0.2% bovine serum albumin.
  • KRH Krebs-Ringers/HEPES
  • the receptor-binding assay was carried out using HEK293T cells stably transfected with insulin or IGF-1 receptor. These were plated in 96-well Isoplate wells (white wall and clear bottom; Perkin-Elmer) coated with poly L-lysine at a density of 80,000 ⁇ 1 ⁇ 8/200 ⁇ well. At the start of the experiment, the medium was aspirated and cells were washed with 250 ⁇ L of phosphate buffered saline.
  • bovine serum albumen (BSA) in binding buffer (20 mM HEPES, 1.5 mM CaCl 2 , 50 mM NaCl, 0.01% NaN 3 ; pH 7.5), and 50 ⁇ of each concentration was added to each well in triplicate.
  • Eu-DTTA insulin or IGF-1 was diluted, and 50 ⁇ , (approx. 100,000 average fluorescent units) was added to each well.
  • the cells were allowed to stand at room temperature for 1 h, after which the cells were washed with 250 ⁇ , of phosphate-buffered saline.
  • the plate was developed for fluorescence measurements by adding 100 ⁇ ⁇ of enhancement solution ( ⁇ -nitrilotriacetic acid [ ⁇ - ⁇ ], trioctylphosphine oxide [TOPO], and 0.1% [w/v] Triton X-100 in 0.1M acetic acid adjusted to pH 3.2) and allowed to stand at room temperature for 45 min, during which Eu was liberated from chelation with DTTA to form a highly fluorescent chelate inside a protective micelle with ⁇ - ⁇ and TOPO.
  • the fluorescence measurement was carried out on a Victor multilabel reader (Perkin-Elmer) using the measurement settings for europium (excitation at 340 nm, emission at 614 nm, delay time of 400 ⁇ , and measurement time of 400 ⁇ ).
  • Binding assays were performed on HEK293 cells transfected with insulin or IGF-1 receptor. Cells were plated in either white or black CoStar 96-well plates at a density of 12,000 cells per well and were allowed to grow for 3 days. On the day of the experiment, media were aspirated from all wells. Then 50 ⁇ of nonlabeled ligand and 50 ⁇ of Eu-labeled ligand were added to each well.
  • Ligands were diluted in binding media (DMEM, ImM 1 ,10-phenanthroline, 200mg/L bacitracin, 0.5mg/L leupeptin, 0.3% BSA), and samples were tested in quadruplicate unless otherwise noted.
  • Cells were incubated in the presence of ligands for 40min at 37 °C. Following the incubation, cells were washed for 3 times with wash buffer (50mM Tris-HCl, 0.2% BSA, 30mM NaCl). Enhancement solution was added (100iL /well), and the plates were incubated for at least 30min at 37 °C prior to reading.

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Abstract

L'invention concerne des peptides marqués au lanthanide, tels que l'insuline marquée au lanthanide, le facteur de croissance insulinique 1 (IGF-1), le facteur de croissance insulinique 2 (IGF-2), le glucagon, le peptide-1 apparenté au glucagon, des dérivés et/ou des analogues de ceux-ci, ainsi que leur utilisation pour mettre au point des dosages biologiques solides.
PCT/CN2013/077729 2013-06-24 2013-06-24 Peptide marqué au lanthanide et utilisation de celui-ci WO2014205617A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107884586A (zh) * 2017-10-31 2018-04-06 吴灿军 一种磁分离均相免疫检测目标蛋白的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783524A (en) * 1985-09-17 1988-11-08 Monsanto Company Growth factors
EP1193272B1 (fr) * 2000-10-02 2004-06-30 Yonsei University Analogues d'insuline à base d'une chaine simple
WO2007081824A2 (fr) * 2006-01-06 2007-07-19 Case Western Reserve University Protéines résistantes à la fibrillation
US7291594B2 (en) * 2002-10-11 2007-11-06 Sanwa Kagaku Kenkyusho Co., Ltd. GLP-1 derivative and preparation thereof absorbable via mucous membrane
US20090137456A1 (en) * 2005-11-07 2009-05-28 Indiana University Research And Technology Glucagon analogs exhibiting physiological solubility and stability
CN101511390A (zh) * 2006-08-31 2009-08-19 弗·哈夫曼-拉罗切有限公司 制备胰岛素样生长因子-1与聚(乙二醇)的偶联物的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783524A (en) * 1985-09-17 1988-11-08 Monsanto Company Growth factors
EP1193272B1 (fr) * 2000-10-02 2004-06-30 Yonsei University Analogues d'insuline à base d'une chaine simple
US7291594B2 (en) * 2002-10-11 2007-11-06 Sanwa Kagaku Kenkyusho Co., Ltd. GLP-1 derivative and preparation thereof absorbable via mucous membrane
US20090137456A1 (en) * 2005-11-07 2009-05-28 Indiana University Research And Technology Glucagon analogs exhibiting physiological solubility and stability
WO2007081824A2 (fr) * 2006-01-06 2007-07-19 Case Western Reserve University Protéines résistantes à la fibrillation
CN101511390A (zh) * 2006-08-31 2009-08-19 弗·哈夫曼-拉罗切有限公司 制备胰岛素样生长因子-1与聚(乙二醇)的偶联物的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FUKUMORI, R. ET AL.: "Plasma concentrations and effects of glucagon-like peptide-1 (7-36) amide in calves before and after weaning", DOMESTIC ANIMAL ENDOCRINOLOGY, vol. 43, 2012, pages 299 - 306 *
SMALL, BRIAN C. ET AL.: "Establishment of a time-resolved fluoroimmunoassay for measuring plasma insulin-like growth factor I (IGF-I) in fish: effect of fasting on plasma concentrations and tissue mRNA expression of IGF-I and growth hormone (GH) in channel catfish (Ictalurus punctatus", DOMESTIC ANIMAL ENDOCRINOLOGY, vol. 28, 2005, pages 202 - 215 *
YAN, XIAOWEN ET AL.: "Absolute Quantification of Intact Proteins via 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic acid-10-Maleimidoethylacetamide-Europium Labeling and HPLC Coupled with Species-Unspecific Isotope Dilution ICPMS", ANALYTICAL CHEMISTRY, vol. 82, no. 4, 15 February 2010 (2010-02-15), pages 1261 - 1269 *
YUAN, JINGLI ET AL.: "A New Tetradentate beta -Diketonate-Europium Chelate That Can Be Covalently Bound to Proteins for Time-Resolved Fluoroimmunoassay", ANALYTICAL CHEMISTRY, vol. 70, no. 3, 1 February 1998 (1998-02-01), pages 596 - 601 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107884586A (zh) * 2017-10-31 2018-04-06 吴灿军 一种磁分离均相免疫检测目标蛋白的方法

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