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Human monoclonal antibody against IgE, preparation method and purpose thereof

A monoclonal antibody, fully human technology, applied in the biological field, can solve the problems of low affinity, failure to achieve full humanization, no data on safety and effectiveness, and achieve the effect of high antibody affinity

Active Publication Date: 2014-05-14
越海百奥药业(绍兴)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The drug is used for adults and adolescents with moderate to severe persistent asthma, but there is no data on the safety and effectiveness of the drug for other allergic states
Xolair is a humanized antibody, which retains the mouse CDR region and a small amount of mouse residues in the FR region, but it has not yet achieved full humanization, and there is a problem of low affinity

Method used

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  • Human monoclonal antibody against IgE, preparation method and purpose thereof
  • Human monoclonal antibody against IgE, preparation method and purpose thereof
  • Human monoclonal antibody against IgE, preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0035] Preparation of Example Antibodies

[0036] (1) Cloning of human antibody light and heavy chain constant region genes

[0037] Lymphocyte separation fluid (product of Dingguo Biotechnology Development Co., Ltd.) was used to separate healthy human lymphocytes, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). Research, 1982, 10: 4071-4079) respectively designed primers using RT-PCR reaction to amplify the antibody heavy chain and light chain constant region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (promega company product). After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 1 and SEQ ID NO: 2 show the heavy chain constant region (C H) nucleotide sequence and amino acid sequence. SEQ ID NO: 3 and SEQ ID NO: 4 show the light chain constant region (C L ) nucleotide sequence and amino acid sequence. The correct clone in thi...

experiment example 1

[0049] Experimental example 1. Affinity detection of anti-IgE antibody 2E19

[0050] Affinity constants of anti-IgE antibodies were detected by surface plasmon resonance (SPR) using a Biacore T100 system (Biacore AB, Uppsala, Sweden). The Fc fragment of the recombinant human IgE antibody was covalently bonded to the CM5 biosensor chip (Biacore) through the amino group, and ① fully human antibody 2E19; ② fully human antibody Xolair (Omalizumab, a commercially available product listed abroad); ③ negative control Antibody Trastuzumab (commercially available product) (in PBS / 0.05% TWEEN-20 (ICI Americas) (detergent) solution) was made into different concentrations (2-fold ratio concentration dilution), and passed through the chip at a flow rate of 50 μl / min. After each check, wash with 5 μl of 50 mM hydrochloric acid aqueous solution at a flow rate of 3 μl / min, thereby the residual antibody is eluted from the immobilized ligand. Using BIAevaluation software (T100evaluation 2.0 ver...

experiment example 2

[0054] Experimental example 2. Anti-IgE antibody 2E19 inhibits the binding experiment of IgE and soluble FcεRI

[0055] Human IgE can specifically bind to its receptor FcεRI. The dose-effect curve of horseradish peroxidase (HRP)-labeled IgE binding to FcεRI should be "S"-shaped, that is, with the increase of HRP-IgE concentration, the degree of color development will gradually increase until it reaches saturation; anti-IgE antibody It can specifically bind to the receptor binding site of IgE and inhibit the binding of IgE to the corresponding receptor FcεRI. If there is anti-IgE antibody competition for HRP-IgE in the reaction system, under the condition of constant HRP-IgE concentration, with the increase of the added anti-IgE concentration, the amount of HRP-IgE bound by FcεRI will gradually decrease, so the dose-effect curve is Reverse "S" shape; IC 50 The smaller the value, the stronger the ability of the added anti-IgE antibody to compete with HRP-IgE, that is, the bett...

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Abstract

The invention relates to the biotechnology field, and more specifically, the invention discloses a human monoclonal antibody against IgE, a preparation method and a purpose thereof. According to the invention, a natural human phage antibody library of large capacity is constructed;; a human antibody 2E19 strain against IgE is obtained through screening from the library; an amino acid sequence of a heavy chain variable region is expressed as SEQ ID NO: 6; and the amino acid sequence of a light chain variable region is expressed as SEQ ID NO: 8. Besides, the invention also discloses the preparation method of the antibody 2E19, as well as a nucleotide sequence for encoding the antibody 2E19 and an expression vector and a host cell comprising the nucleotide sequence. The antibody 2E19 of the invention has a higher antibody affinity, so as to be able to obviously inhibit degranulation caused by the combination of IgE and FC epsilon RI on a cell surface, and the antibody 2E19 does not combine with the IgE combined with the FC epsilon RI on the cell surface. The antibody 2E19 of the invention could be used for the preparation of a medicine for treating hypersensitivity diseases, especially for treating allergic asthma.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention discloses a fully human monoclonal antibody, its preparation method and application. Background technique [0002] Hypersensitivity reaction refers to an abnormal or pathological immune response produced by the body after being stimulated by a certain antigenic substance. It often manifests as increased immune reactivity, and it often occurs when the body is exposed to the same antigen again. Hypersensitivity and immune response are essentially the body's specific immune response to certain antigenic substances, but the former mainly manifests as tissue damage and (or) physiological dysfunction, while the latter mainly manifests as physiological defense effects. [0003] There are many classification methods for hypersensitivity reactions, but most of them currently use the classification of Gell and Coombs (1963), which is divided into four types I to IV accordin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/42C12N15/13C12N15/85C12N5/10A61K39/395A61P37/08A61P11/06
Inventor 聂丽王淑蕙郭怀祖
Owner 越海百奥药业(绍兴)有限公司
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