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Humanized anti-A[beta] monoclonal antibody and application thereof

A monoclonal antibody, humanized technology, applied in the direction of antibodies, applications, chemical instruments and methods, etc.

Active Publication Date: 2020-08-11
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0030] Although there is a certain amount of theoretical knowledge in the field of treatment and prevention of Alzheimer's disease, there is still a need to improve compositions and methods for the treatment and / or prevention of this disease, and there is a need for antibodies and treatments that can target Aβ

Method used

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  • Humanized anti-A[beta] monoclonal antibody and application thereof
  • Humanized anti-A[beta] monoclonal antibody and application thereof
  • Humanized anti-A[beta] monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Example 1 Preparation of Aβ antigen and positive control antibody

[0168] Preparation of Aβ monomer and polymer mixture

[0169] Aβ 1-42 , Aβ 1-16 , Aβ 14-29 The peptide was synthesized by Gill Biochemical (Shanghai) Co., Ltd. Aβ 1-42 The amino acid sequence of the polypeptide is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID: 35), Aβ 1-1 The amino acid sequence of the 6 polypeptides is: DAEFRHDSGYEVHHQK (SEQ ID: 36), Aβ 14-29 The amino acid sequence of the polypeptide is: HQKLVFFAEDVGSNKGA (SEQID: 37).

[0170] Aβ 1-42 Monomer (Aβ monomer for short) preparation method: to 1mgAβ 1-42 Add 1ml of hexafluoroisopropanol (HFIP) to the dry peptide powder, vortex for 1min, and sonicate in a water bath for 1-5min until completely dissolved; place in a shaking incubator at 37°C and 200rpm for 1.5h; use a vacuum rotary dryer to evaporate completely Hexafluoroisopropanol; to dry Aβ 1-42 Add 192 μl of anhydrous dimethyl sulfoxide (DMSO) to the polypeptide to dissolv...

Embodiment 2

[0187] Example 2 Preparation of anti-Aβ monoclonal hybridoma

[0188] Immunization of BALB / c mice

[0189] The Aβ1-42 polypeptide antigen and Freund's complete adjuvant were vortexed and mixed according to the dosage, and after complete emulsification, 6-week-old BALB / c female mice were immunized for the first time. Each mouse was intraperitoneally injected with 200 μg antigen, and 3 groups of mice were immunized with 5 mice in each group. Two weeks after the first immunization, the mice were immunized intraperitoneally for the second time, using Freund's incomplete adjuvant, and the dose of immunizing antigen was the same as the first immunization. Thereafter, the mice were intraperitoneally immunized twice a month with the same dose of adjuvant and antigen as the second immunization.

[0190] After the first immunization, a small amount of orbital blood was collected from the mice every six weeks and the serum titer was tested. After the serum titer was detected by indirec...

Embodiment 3

[0230] Example 3 Detection of Anti-Aβ Monoclonal Antibody Inhibiting Aβ Aggregation

[0231] Dissolve Aβ dry powder to 1 mg / ml with 8.2% DMSO / DPBS solution (DMSO: sigma; DPBS: Hyclone), dilute Aβ solution to 33 μg / ml with DPBS, anti-Aβ monoclonal antibody 066-4.6.8, 066-4.18.2, 066-4.22.1, 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2 diluted to 450μg / ml (IC100), Dilute ThT(sigma) to 20 μM with ultrapure water. Take 50 μl of antibody dilution and add it to a 96-well black plate (corning), then add 50 μl of Aβ dilution to it, and finally add 100 μl of ThT, incubate at room temperature in the dark for 24 hours, and detect the fluorescence intensity with a multifunctional microplate reader (Ex / Em=440 / 485). The abscissa represents different sample addition groups, and the ordinate represents the relative fluorescence intensity. The results are shown in Figure 3. In Figure 3 (A), when the relative fluorescence intensity of the IgG group was 1.0, the relativ...

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Abstract

The invention relates to the technical field of antibody drugs, in particular to a humanized anti-A[beta] monoclonal antibody and an application thereof. The humanized anti-A[beta] monoclonal antibodyprovided by the invention can inhibit A[beta] monomer polymerization and protect nerve cells from being infected with A[beta] toxicity, has a certain effect of improving cognitive learning and memoryability of Alzheimer dementia model mice, and can be used for treatment and diagnosis of diseases and symptoms related to amyloidosis, such as the Alzheimer disease.

Description

technical field [0001] The invention relates to the technical field of antibody medicines, in particular to humanized anti-Aβ monoclonal antibodies and applications thereof. Background technique [0002] Aβ [0003] Amyloid β (Aβ) is encoded by human chromosome 21 gene, contains 39-43 amino acids, has a β-sheet structure, is hydrophobic, and has a molecular weight of 4KDa. Aβ is derived from the residue polypeptide produced by the cleavage of amyloid precursor protein (APP) by proteolytic enzymes. APP can be decomposed by α-, β-, γ-protease, and the decomposed products have different biological functions. Among them, the continuous action of β-protease and γ-protease produces Aβ. The γ-protease generates the C-terminus of Aβ, cleaves the transmembrane region of APP and can then generate a large isoform of 39-43 amino acid residues. The most common of all residue subtypes are Aβ40 and Aβ42. The former is typically formed when the endoplasmic reticulum is severed, while ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70G01N33/68G01N33/577A61K39/395A61P25/28
CPCC07K16/18C12N15/70G01N33/68G01N33/577A61P25/28G01N2800/2821A61K2039/505C07K2317/30C07K2317/76C07K2317/70C07K2317/24C07K2317/92G01N33/6896G01N2333/4709A61K47/6843C07K2317/52C07K2317/565C07K2317/567
Inventor 冯晓梁阳秋金磊孙大为王涛肖亮刘爽陈宇珩李正一
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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