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Hinge region modification body of human IgG2 antibody

A hinge region and antibody technology, applied in the direction of antibodies, cells modified by introducing foreign genetic material, anti-animal/human immunoglobulin, etc., can solve the problems of increasing the sensitivity and reducing stability of IgG2 antibodies

Active Publication Date: 2015-03-11
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These potential protease hydrolysis sites increase the sensitivity of IgG2 antibodies to protease hydrolysis and reduce stability

Method used

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  • Hinge region modification body of human IgG2 antibody
  • Hinge region modification body of human IgG2 antibody
  • Hinge region modification body of human IgG2 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, wild-type Denosumab and AB4 and antigen-binding affinity determination

[0046] Firstly, the binding ability of HRP-labeled Denosumab antibody to antigen was detected by direct ELISA method. First, horseradish peroxidase (HRP, Boehringer Ingelheim)-labeled anti-human RANKL antibody Denosumab (Amgen) was used as a reagent (350 μg / mL), and serially diluted in proportion. The antigen RANKL-mFc (100 μl, 0.5 μg / mL) fused with mouse IgG Fc was coated on a microtiter plate (Corning) and left overnight at room temperature. The coating solution was discarded, each well was blocked with skimmed milk dissolved in PBS for 0.5 hour, and then washed with PBS containing 0.05% Tween-20. Then 50 μl of HRP-labeled antibody Denosumab at different concentrations was added to each well, and after reacting for 1 hour, the wells were washed with PBS containing 0.05% Tween-20. Finally, the OD value of each well was read with a microplate reader (Thermo Fisher) at dual wavelengt...

Embodiment 2

[0048] Example 2. Competitive binding assay with wild-type Denosumab antibody

[0049] HRP-labeled anti-human RANKL antibody Denosumab was used as a reagent (initial concentration 350 μg / mL, diluted 1:1000). Coat the microtiter plate with the antigen RANKL-mFc (100 μl, 0.5 μg / mL) fused with mouse IgG Fc and leave overnight at room temperature. The coating solution was discarded, each well was blocked with skimmed milk dissolved in PBS for 0.5 hour, and then washed with PBS containing 0.05% Tween Tween-20. Then, a mixture of 50 μl AB4 and 50 μl HRP-labeled antibody Denosumab (250ng / mL) was added to each well, and after reacting for 1 hour, the wells were washed with PBS containing 0.05% Tween-20 (Tween-20). Unlabeled Denosumab and an unrelated antibody against another antigen were used as positive and negative controls. The initial concentration of the test antibody and control antibody was 30 μg / mL, and then serially diluted at a ratio of 1:2. Finally, read the OD value of e...

Embodiment 3

[0050] Example 3, RP-HPLC analysis of heterogeneity between wild-type Denosumab antibody and AB4 antibody

[0051] RP-HPLC was used to analyze the heterogeneity caused by the disulfide bond between the AB4 antibody and the control antibody, that is, the wild-type Denosumab antibody. Wufeng HPLC LC100 high performance liquid chromatograph is used, equipped with P100 high pressure constant flow pump, UV100 ultraviolet detector and WS100 chromatographic workstation.

[0052] Molecular sieve HPLC chromatography conditions: gel chromatography column TSK gel G3000 SWXL (7.8mm×300mm) (TOSOH Company, Cat No. 0008541); mobile phase: 100mM sodium phosphate, 500mM sodium chloride, 5% ethanol, pH 7.0; flow rate: 0.5mL / min; column temperature: 25°C; injection volume: 20μl.

[0053] RP-HPLC conditions: Sephasil Peptide C8 chromatography column (4.6mm×250mm, 5μm, Pharmacia Biotech); mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 70% isopropanol, 20% acetonitrile...

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Abstract

The invention discloses a hinge region modified recombinant human IgG2 antibody. Amino acids at the sites of Glu216Arg217Lys218 are deleted in a heavy chain hinge region of the antibody, and amino acid is substituted and / or deleted at the site of Cys219 and / or Cys220. The invention further provides a method for improving the antiprotease hydrolysis effect of the recombinant human IgG2 antibody. According to the IgG2 antibody provided by the invention, the heterogeneity caused by disulfide bonds in the hinge region of the antibody is eliminated, and the effect of improving the antiprotease hydrolysis effect is achieved.

Description

technical field [0001] The present invention relates to the field of antibodies, more specifically, to a hinge region modification of a recombinant human IgG2 antibody, in which amino acid residues at specific positions in the hinge region are deleted and / or replaced. Background technique [0002] When developing antibody-based pharmaceuticals, their physical properties, especially homogeneity and stability, are extremely important. Regarding IgG2 subtypes, heterogeneity due to disulfide bonds in the antibody hinge region has been reported. For example, Denosumab, a fully human monoclonal antibody targeting RANKL (receptor activator of nuclear factor κB ligand) developed by Amgen, is an IgG2 antibody. Like many natural IgG2 subtype antibodies isolated from plasma, it has three different forms of isoforms IgG2-A, IgG2-A / B and IgG2-B, resulting in heterogeneity of the final product (Wypych J et al., J Biol Chem , 2008, 283:29266-29272). The pairing mode, mismatch or incompl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/10A61K39/395A61P19/08A61P19/10
Inventor 李强李媛丽孙见宇周若云孙乃超
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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