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Site-specific radioisotope-labeled antibody using IgG-binding peptide

A radioactive and nuclide technology, applied in in vivo radioactive preparations, preparations for in vivo experiments, peptides, etc., can solve the problems of reduced antibody function and high development costs

Pending Publication Date: 2018-12-21
KAGOSHIMA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This site-specific antibody modification technology is being developed, but in many cases, it is necessary to engineer the antibody itself, but considering the reduced function of the modified antibody and the high development cost, it may not be a useful method.

Method used

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  • Site-specific radioisotope-labeled antibody using IgG-binding peptide
  • Site-specific radioisotope-labeled antibody using IgG-binding peptide
  • Site-specific radioisotope-labeled antibody using IgG-binding peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0310] [Example 1: X-ray crystal structure analysis of the complex of IgG-binding peptide and IgG]

[0311]

[0312] (1) Preparation of IgG binding peptide solution

[0313] By utilizing the peptide solid-phase synthesis method of the F-moc method, a sequence (SEQ ID NO: 31, wherein HC is homocysteine, 4th 2 Cys at position 1 and 14, and 2 homocysteine ​​at position 2 and 16 respectively form a disulfide bond in the molecule) cyclic homocysteine ​​peptide. 0.8 mg of the prepared IgG-binding peptide powder was dissolved in 24 µL of 100% dimethylsulfoxide (Wako Pure Chemical Industries, Ltd.) to prepare an IgG-binding peptide solution.

[0314] (2) Preparation of a complex of Fc and IgG-binding peptide

[0315] In 20 mmol / L phosphate buffer (pH 7.0) containing 10 mM EDTA and 1 mM L-cysteine, the hinge of human IgG (Chugai Pharmaceutical) was digested with papain (manufactured by Roche) at 37 °C. )part. Next, using a cation exchange chromatography column (TSKgel SP5-PW (Tos...

Embodiment 2

[0326] [Example 2: Preparation and properties of peptides for labeling]

[0327]

[0328] Using the Fmoc solid-phase synthesis method and according to conventional methods, synthesize amino-PEG4 modified amino-PEG4 with biotin (Biotin) or 5 / 6-TAMURA succinimidyl ester (AnaSpec company) (fluorescent pigment) The peptide GPDCAYHXGELVWCTFH (SEQ ID NO: 2) was synthesized (where the C-terminus is amidated). After removal of the protecting group, intramolecular S-S bond formation was performed under oxidative conditions in aqueous solution at pH 8.5 using reverse phase HPLC at a flow rate of 1.0 ml / min through a gradient of 10% to 60% acetonitrile containing 0.1% TFA Elution, purification of peptides with intramolecular S-S bonds.

[0329] 100 µL of a DMF solution containing 1 mM of the purified IgG-binding peptide was mixed with 100 µL of an acetonitrile solution of 100 mM DSS or DSG (Thermo Fisher Scientific), and reacted overnight at room temperature. After diluting the react...

Embodiment 3

[0338] [Example 3: Specific modification of human IgG-Fc using IgG-binding peptide]

[0339]

[0340] A labeling reagent peptide modified with DSS or DSG to an IgG-binding peptide (type I (R8K)) with biotin-PEG4 added to the N-terminus was prepared by the same method as in Example 2, and reacted with human IgG Fc to examine human Labeling reaction of IgG Fc. That is, after purifying the IgG-binding peptide (R8K) (200 pmol / 5 μL in 0.1% TFA) reacted with excess DSS or DSG using a reverse-phase column by the same method as in Example 2, acetonitrile was removed under reduced pressure, and then about 1 / 8 of 0.5M Na 2 HPO 4 For neutralization, immediately add protein samples (hIgG (Chuwai Pharmaceutical), hIgA (Athens Research & Technology), HSA (Sigma-Aldrich), or serum (collected from healthy people)) at a molar ratio of 10 times (each 40pmol / 5μL, Serum was diluted 10-fold with PBS), and the final volume was adjusted to 20 μL with PBS, and then left at room temperature for 5...

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Abstract

The present invention pertains to: an IgG-binding peptide containing a ligand which can be bound to a radioactive metal nuclide; an IgG-binding peptide labeled with a radioactive metal nuclide; a complex of the IgG-binding peptide with IgG; and a nuclear medical imaging diagnostic agent or a cancer diagnostic agent, etc., containing the IgG-binding peptide or the complex.

Description

technical field [0001] The present invention relates to an IgG-binding peptide containing a ligand capable of binding to a radioactive metal nuclide, an IgG-binding peptide labeled with a radioactive metal nuclide, a complex of the IgG-binding peptide and IgG, and a compound containing the IgG-binding peptide or complex Nuclear medicine imaging diagnostic agent or cancer diagnostic agent, etc. Background technique [0002] Antibodies have been used to detect target molecules in various research and development, and they are also very important in the industry as detection reagents and diagnostic drugs. In addition, antibodies are also attracting attention as drugs for disease treatment due to their specificity for target molecules. [0003] In order to add functions to antibodies, radioactive isotope labeling by iodination, addition of chelate compounds (Non-Patent Document 1) and the like are available. Now these modifications are mainly carried out through the amino grou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K51/00C07K7/64C07K16/00G01N33/534G01N33/574
CPCC07K16/00C07K7/08C07K7/64G01N33/534G01N33/574A61K51/088A61K51/1051A61K51/1093C07K7/06A61K2123/00
Inventor 伊东祐二正山祥生林明希男中田德仁
Owner KAGOSHIMA UNIV
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