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Specific modification of antibody by IgG-binding peptide

A residue and amino acid technology, applied in the field of production of the complex, can solve the problems of reaction yield impact, complex reaction steps, high development cost, etc.

Active Publication Date: 2018-01-19
KAGOSHIMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it has been reported to use transglutaminase (TG) to modify a specific glutamine naturally or artificially introduced in an antibody (Non-Patent Documents 12 to 13), and it is known that depending on the structure of the introduced compound And the steric environment of the glutamine residue as the target greatly affects the reaction yield
Furthermore, there are modification techniques using sugar chains on antibody Fc as targets (Non-Patent Documents 14 to 15). Although these methods are site-specific, there is a problem that the reaction steps are complicated because oxidation modification of sugar chains is required.
Such site-specific antibody modification technology is being developed, but in many cases, it is necessary to engineer the antibody itself, and this may not be a useful method in view of the reduction in antibody function and high development costs associated with the change.

Method used

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  • Specific modification of antibody by IgG-binding peptide
  • Specific modification of antibody by IgG-binding peptide
  • Specific modification of antibody by IgG-binding peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0374] [Example 1: Analysis of X-ray crystal structure of a complex of IgG-binding peptide and IgG]

[0375]

[0376] (1) Preparation of IgG binding peptide solution

[0377] By using the peptide solid-phase synthesis method of the F-moc method, according to the conventional method to prepare the G(HC)DCAYHRGELVWCT(HC)H-NH 2 The sequence (SEQ ID NO:31, wherein, HC is homocysteine, the 4th and 14th two Cys, the 2nd and the 16th two homocysteine ​​respectively form two Sulfur bond) 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.

[0378] (2) Preparation of a complex of Fc and IgG-binding peptide

[0379] 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, the human...

Embodiment 2

[0390] [Example 2: Preparation and properties of peptides for labeling]

[0391]

[0392] Use biotin (Biotin) or 5 / 6-Tamura succinimidyl ester (TAMURA succinimidyl ester) (AnaSpec company) (fluorochrome) to amino-PEG4 synthesis peptide GPDCAYHXGELVWCTFH (SEQ IDNO:2) (wherein, The C-terminus is amidated) was synthesized according to a conventional method using Fmoc solid-phase synthesis. 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 purifies peptides with intramolecular S-S bonds.

[0393]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 reactant to 2.5 times with 0.1...

Embodiment 3

[0400] [Example 3: Specific modification of human IgG-Fc using IgG-binding peptide]

[0401]

[0402] 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 study 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 reversed-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 after making the final volume to 20 μL with PBS, it was left at room temperature for 5 mi...

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PUM

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Abstract

The present invention pertains to an IgG-binding peptide, an IgG-binding peptide modified with a cross-linking agent, a complex of IgG and said IgG-binding peptide modified with a cross-linking agent,and a method for preparing said complex.

Description

technical field [0001] The present invention relates to an IgG-binding peptide, an IgG-binding peptide modified with a cross-linking agent, a complex of the IgG-binding peptide modified with a cross-linking agent and IgG, a method for producing the complex, and the like. Background technique [0002] Antibodies are widely used in the detection of target molecules in various research and development at present, and 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 from the viewpoint of specificity for their target molecules. [0003] As chemical modifications for adding functions to antibodies, there are enzymes such as alkaline phosphatase (AP) and peroxidase (HRP) (Non-Patent Documents 1 and 2), and iodide and chelate for radioactive isotopes. compound (Non-Patent Document 3), and modification with low-molecular-weight compounds such as biotin (Non-Patent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K39/395C07K16/00C07K19/00
CPCC07K16/00C07K7/08A61K38/00A61K47/00C07K2319/00C07K14/4748A61K47/62A61K39/395C07K19/00A61K47/64
Inventor 伊东祐二
Owner KAGOSHIMA UNIV
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