Targeting EGFR and HER2 double-specific antibody and application thereof

A bispecific antibody and antibody technology, applied in the direction of application, antibody, specific peptide, etc., can solve the problems of easy tolerance and inability to exert therapeutic effect for a long time, and achieve the effect of reducing the amount of antibody, having a significant killing effect, and a remarkable effect

Inactive Publication Date: 2017-11-07
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, small molecule inhibitors are prone to tole

Method used

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  • Targeting EGFR and HER2 double-specific antibody and application thereof
  • Targeting EGFR and HER2 double-specific antibody and application thereof
  • Targeting EGFR and HER2 double-specific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, recombinant plasmid construction

[0039] All experimental operations in plasmid construction are referred to "Molecular Cloning Experiment Guide" (Second Edition, Science Press). The DNA molecules shown in the sequences 1, 3, 5 and 7 of the sequence listing were respectively inserted into the pZJC vector to obtain 4 kinds of recombinant plasmids. Using the heavy and light chains of the humanized cetuximab antibody and Herceptinumab as templates, the corresponding site mutations and fragment swaps were introduced, and primers were designed for amplification. The corresponding primers are shown in Table 1 below:

[0040] Table 1 Synthetic Crossmab structural gene primer sequence

[0041]

Embodiment 2

[0042] Example 2, preparation of bispecific antibodies

[0043] (1) Culture well-growing HEK293F cells into 125ml shake flasks, and adjust the number of cells to 5×10 the day before transfection 5 / ml, the activity reaches more than 90%, and the medium is 30ml. Cultivate at 37°C and 125rpm.

[0044] (2) Measure the cell number and viability on the day of transfection, and the viability should be greater than 95%. Replace with fresh Freestyle293F medium after centrifugation. Take 30μg combined plasmid DNA in Opti-MEM medium, the total volume is 1ml.

[0045] (3) Add 60 μl of 293F liposomes into Opti-MEM medium, the total volume is 1 ml.

[0046] (4) Incubate at room temperature for 5 minutes, slowly add DNA into the diluted liposomes, gently blow and mix with a pipette tip, and let stand at room temperature for 20-30 minutes. After incubation, mix 2ml of liposomes and plasmids solution was added to the cell suspension.

[0047] (5) Continue to culture in a shaker at 37° C...

Embodiment 3

[0054] Embodiment 3, SDS-PAGE detection

[0055] (1) Sample treatment: add three times the volume of the sample to one volume of 4×Loading Buffer (Loading Buffer is divided into denatured reduction and denatured non-reduced types), mix well, boil for 10 min, centrifuge at 12000 rpm for 10 min, and set aside.

[0056] (2) Glue compounding: First prepare the separation gel, add isopropanol on top of the separation gel, let it stand at room temperature for about 30 minutes until the gel solidifies, and pour off the isopropanol. Then prepare concentrated gel, add the concentrated gel and insert it into the comb mold quickly, let it stand at room temperature for 30 minutes until the gel solidifies, and pull out the comb mold.

[0057] (3) Sample loading: Use a small pipette tip to draw the sample for sample loading, load 5 μl of the sample on the Marker, and supplement the gel holes on both sides with 1×LoadingBuffer.

[0058] (4) Electrophoresis: The protein electrophoresis buffe...

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Abstract

The invention discloses a targeting EGFR and HER2 double-specific antibody and an application thereof. An antigen bonding zone thereof is composed of humanized cetuximab and herceptin. According to the invention, a bioengineering technology is utilized to construct a double-specific antibody for simultaneously targeting and combining with EGFR and HER2 antigens. The invention provides DNA sequences of two heavy chains and two light chains of the antibody and expression host cells thereof. The invention also protects the application of the double-specific antibody for preparing drugs for non-small cell lung cancers. The double-specific antibody has an important application value for treating the non-small cell lung cancers.

Description

technical field [0001] The present invention mainly relates to a bispecific antibody targeting EGFR and HER2 and its application. Background technique [0002] Cancer is a serious threat to human health, especially in patients with malignant tumors, whose condition develops rapidly, is prone to metastasis, and has a high mortality rate. At present, it ranks second in the statistics of deaths caused by various diseases. In recent years, with the aggravation of environmental pollution and other factors, the incidence of tumors has increased significantly. However, conventional treatments such as radiotherapy, chemotherapy, and surgery can alleviate the disease to a certain extent and improve the survival time, but there are also many disadvantages, such as large side effects and easy recurrence. The therapeutic effect is difficult to be improved further. [0003] Among various cancers, lung cancer is the malignant tumor with the fastest increasing morbidity and mortality. ...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/85A61K39/395A61P35/00
CPCC07K16/2863C07K16/32C07K2317/24C07K2317/31C07K2317/73C07K2317/92C12N15/85
Inventor 张部昌周琴徐昌志韩倩倩王相芳鲁亚芳喻阳邵国建
Owner ANHUI UNIVERSITY
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