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Anti-HER3 antibody, preparing method thereof and applications of the antibody

A technology of antibody and antibody heavy chain, which is applied in the field of biomedicine and can solve problems such as lack of

Active Publication Date: 2016-03-02
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of a highly sensitive and specific antibody against HER3 in this field

Method used

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  • Anti-HER3 antibody, preparing method thereof and applications of the antibody
  • Anti-HER3 antibody, preparing method thereof and applications of the antibody
  • Anti-HER3 antibody, preparing method thereof and applications of the antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1, Preparation of HER3 mouse monoclonal antibody

[0153] 1. Preparation of HER3 monoclonal antibody hybridoma cell lines

[0154] 1. Immunogen

[0155] The immunogen is the full-length phenylalanine extracellular region (HER3-ECD-H584F) whose histidine at position 584 in the extracellular region of HER3 is mutated by CHO / dhfr - Cells (purchased from ATCC) were stably expressed, and the supernatant of cell culture was purified.

[0156] 2. Immunization of Balb / c mice

[0157] Balb / c mice were purchased from Shanghai Slack Co., Ltd. All Balb / c mice used for immunization were 3-week-old, female, standardized disease-free, healthy purebred mice, which met the US FDA standards.

[0158] 3. Balb / c mouse immunization method

[0159] The first immunization: Mix 50 μg (250 μl) antigen with 250 μl MF59 adjuvant, prepare a 500 μl solution, and inject it into Balb / c mice at multiple points and paws subcutaneously. The second immunization: three weeks after the first...

Embodiment 2

[0178] Embodiment 2, mouse monoclonal antibody affinity determination

[0179] The affinity of the HER3 monoclonal antibody was determined using the proteonTMXPR36ProteinInteractionArraySystem method (refer to BronnerV, DenkbergG, PeledM, et al. Therapeutic antibodies: Discovery and development using the ProteOnXPR36biosensorinteractionarraysystem. AnalBiochem2010; 406: 147-156).

[0180] Antibody number

Embodiment 3

[0181] Example 3. Recognition of Her3 molecules on cell surface by anti-Her3 monoclonal antibody

[0182] Flow cytometry (FACS) was used for detection and indirect immunofluorescence staining.

[0183] (1) Counting: Take SKBR-3 cells in logarithmic growth phase, and adjust the concentration of single cell suspension to 2×106 / ml.

[0184] (2) Washing: take 1ml of single cell suspension and add it into a 1.5ml centrifuge tube, 1000rpm×3min. Discard the supernatant, wash and resuspend the cells with 2% PBA (PBS plus 2% fetal bovine serum), 1000 rpm×3min, discard the supernatant.

[0185] (3) Primary antibody incubation: dilute the anti-Her2 monoclonal antibody to 10 μg / ml with 2% PBA, add 200 μl, mix the cells by gently pipetting, and place in an ice bath at 4°C for 30 minutes. At the same time, a blank control and a mouse IgG monoclonal antibody isotype control were made. 1000rpm×3min, discard the supernatant.

[0186] (4) Fluorescent secondary antibody incubation: wash on...

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Abstract

The invention provides an anti-HER3 antibody, a preparing method thereof and applications of the antibody and particularly provides a novel anti-HER3 antibody. The antibody is high in affinity with HER3 molecules and can specifically combine an antigen molecule. In particular, a human-mouse chimeric antibody effectively reduces immunogenicity of a mouse antibody. By combined application of the anti-HER3 antibody and cetuximab, activity of the cetuximab is significantly improved, and the using amount of the cetuximab is reduced.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to an anti-HER3 antibody, its preparation method and application. Background technique [0002] HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, SEQ ID NO.: 41) is a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family encoding A member of this family also includes HER1 (also known as EGFR), HER2, and HER4, among others. The structure of HER3 includes an extracellular region that binds ligands, an alpha-helical transmembrane region, an intracellular region with a tyrosine kinase domain and a tyrosine-rich C-terminal phosphorylation site. Among them, the extracellular domain can be divided into I (L1), II (S1), III (L2) and IV (S2). Domains I and III are the ligand-binding domains, while domains II and IV are the domains after ligand binding. A conformational change exposes the dimerization domain. Heregulin ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N5/00C12P21/08C12P21/02A61K39/395A61P35/00A61P37/06G01N33/68G01N33/577
Inventor 瞿爱东李翱翔梁红远祝婧烨吴丽娜黄海武陆瑾赵鑫
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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