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Specific binding protein and application thereof

A composition and monoclonal antibody technology, applied in the medical field, can solve problems such as low EGFRvIII specificity and unsatisfactory antibodies

Active Publication Date: 2009-12-16
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, although a variety of antibodies against EGFR antigens have been obtained at home and abroad, these antibodies are often not ideal, such as no or low specificity against EGFRvIII

Method used

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  • Specific binding protein and application thereof
  • Specific binding protein and application thereof
  • Specific binding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] The preparation of embodiment 1 antigen

[0086] 1.1 Prokaryotic expression and purification of the extracellular domain of EGFRvIII protein

[0087] 1.1.1 Vector construction and identification

[0088] Using pLNRNL (encoding full-length EGFRvIII, purchased from Ludwig Institute, San Diego, CA) as a template, the amplification product of EGFRvIIIex with restriction sites BamHI and SalI at both ends was obtained by PCR method, and double-digested with BamHI and SalI, Get the target fragment. The commercially available vector pET28a (available from Novagen) was digested with BglII and SalI, the target fragment was recovered after agarose gel electrophoresis, ligated under the action of T4 ligase to form the vector pET28a-EGFRvIIIex, and then transformed into the commercially available large intestine Bacillus TOP10 (available from Invitrogen) was screened by Kana resistance, and positive clones containing insert fragments were identified by BglII and SalI digestion.

...

Embodiment 2

[0110] Example 2. Antigen immunization and hybridoma screening

[0111] 2.1 Immunity

[0112] (1) Recombinant protein immunization:

[0113] The EGFRvIII extracellular region recombinant protein was fully emulsified and mixed with an equal amount of complete Freund's adjuvant (Sigma) to subcutaneously immunize 6-week-old BALB / c mice, 100 μg per mouse. Four weeks later, the recombinant antigen was emulsified and mixed with incomplete Freund's adjuvant, and the mice were immunized by intraperitoneal injection, 50 μg per mouse, and the intraperitoneal booster immunization was continued at intervals of 2 weeks thereafter. One week after the fourth booster immunization, the recombinant antigen was coated, and the antiserum titer of the mouse was detected by ELISA method > 10 5 .

[0114] (3) Intrasplenic injection to boost immunity:

[0115] Three weeks after the last boost, 20 μg of recombinant antigen was immunized intrasplenicly.

[0116] 2.2 Establishment of hybridoma cell...

Embodiment 3

[0128] Embodiment 3 Detection of binding ability of monoclonal antibody

[0129] 3.1 FACS analysis of receptor binding specificity of 12H23

[0130] Vector build:

[0131] Cells: U87 cells (glioma cell line, normal expression of EGFR, can be purchased from ATCC cell bank), U87-EGFRvIII cells (U87 cell line transfected with pLERNL vector) and A431 cells (human epidermal squamous cell carcinoma, Overexpression of EGFR, available from ATCC cell bank)

[0132] Antibody: the antibody 12H23 prepared in Example 2, and the commercially available C225 monoclonal antibody (as a control). The antibody concentration was 2mg / ml, diluted 1:100.

[0133] 1) The cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of about 90%, and cultured overnight in a 37°C incubator.

[0134] 2) The next day, digest the cells with 10 mM EDTA, centrifuge at 5000 rpm for 3 min, and collect the cells in a 2 ml Eppendorf tube.

[0135] 3) Cells were resuspended in 0.5-1m...

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Abstract

The invention relates to a specific binding protein and an application thereof and particularly provides a monoclonal antibody which can be effectively bound with an epidermal growth factor receptor mutant III (EGFRv III) or partially bound with an epidermal growth factor receptor (EGFR) with overexpressed cells, but has no binding effect with an EGFR with normally expressed cells. In addition, the monoclonal antibody has obvious therapeutic effect on a tumor cell line which expresses the EGFRv III. The invention also provides a method for preparing the monoclonal antibody and a medical composition containing the monoclonal antibody.

Description

technical field [0001] The present invention relates to the field of medicine. More specifically, the present invention relates to specific monoclonal antibodies against epidermal growth factor receptor mutant III (EGFRvIII) and applications thereof. The antibody of the present invention can effectively bind to EGFRvIII or partially bind to epidermal growth factor receptor (EGFR) overexpressed by cells, and the antibody has no binding effect on EGFR normally expressed by cells. The antibodies of the invention can be used to treat tumor cell lines expressing EGFRvIII. Background technique [0002] Epidermal growth factor receptor (EGFR) is the 170 kilodalton membrane glycoprotein product of the proto-oncogene c-erb B (1) . The EGFR gene is the cellular homologue of the erb B oncogene originally identified in avian erythrocytosis virus (1，2) . Activation of this oncogene by gene amplification has been observed in various human tumors (3-6) . [0003] It has been shown i...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
Inventor 李宗海王华茂蒋华石必枝顾健人杨胜利
Owner SHANGHAI INST OF ONCOLOGY
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