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Anti-EGFR (epidermal growth factor receptor) and anti-CD3 (cluster of differentiation 3) bispecific antibody and application thereof

A bispecific antibody and antibody technology, applied in the direction of antibody, application, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems that need to be studied, and achieve the reduction of drug frequency, high affinity, pain relieving effect

Active Publication Date: 2017-05-10
BEIJING DONGFANG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing technology, there is no EGFR and CD3 bispecific antibody drug pro

Method used

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  • Anti-EGFR (epidermal growth factor receptor) and anti-CD3 (cluster of differentiation 3) bispecific antibody and application thereof
  • Anti-EGFR (epidermal growth factor receptor) and anti-CD3 (cluster of differentiation 3) bispecific antibody and application thereof
  • Anti-EGFR (epidermal growth factor receptor) and anti-CD3 (cluster of differentiation 3) bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0061] Specific example 1, the construction of the expression vector of bispecific antibody molecule

[0062] a) Construction of double-specific transient expression vector

[0063] according to figure 1 Design the corresponding gene sequence in the form of bispecific antibody. PTSE was chosen as the expression vector to clone and express the anti-EGFR light chain gene and anti-EGFR heavy chain-CD3 ScFV fusion gene. The light chain gene and the fusion gene were respectively added Sall and BamH1 restriction sites on both sides of the coding region sequence, and the two genes were synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd. and cloned into the PUC19 vector respectively. The two plasmids were respectively transformed into TOP competent (Huitian Dongfang, Cat. No. HT702-03), and the small extraction was carried out with the Kangwei Century Mini Kit (Cat. No. CW0500). and BamHI-HF enzyme digestion, and homologous recombination were performed to obtain express...

specific Embodiment 2

[0066] Specific Example 2, Expression and Purification of Bispecific Antibody Molecules

[0067] a) Expression of tetravalent antibodies

[0068] The endotoxin-free large-scale extraction kit (Kangwei Century, CW2104) was used for large-scale extraction of plasmids, and the specific operation steps were operated according to the instructions provided by the kit.

[0069] Human embryonic kidney cells (HEK293ES suspension cells) were cultured in FreeStyle 293Expression Medium (Gibco, 12338-026), and the cells were passaged every one to two days. After passage, the initial cell density was maintained at 0.2-0.6×10 6 / ml, the cell culture volume is 15~35% of the shake flask volume, and the cell culture flask is placed on a shaker (shaker speed: 135rpm, temperature: 37°C, CO 2 :5%). The day before transfection, HEK293ES cells in logarithmic growth phase and in good growth state were passaged to a cell density of 0.5×10 6 / ml, on a shaker (135rpm, 37°C, 5% CO 2) were cultured ov...

specific Embodiment 3

[0073] Specific Example 3: Binding of bispecific antibody molecules to CD3 and EGFR molecules

[0074] Coat the extracellular region of EGFR or the dimer of the extracellular region of CD3E and CD3G subunits with pH 9.6 carbonate buffer, 100ng / well / 100ul, overnight at 4°C. Wash five times with 300ul / well of 0.1% PBS (PBS-T) buffer solution, then add 1% BSA-PBS solution to block at room temperature for 2h. Add different dilutions of bispecific antibodies or corresponding antibodies. The highest concentration of various bispecific antibodies (or antibodies) is 1uM, and 10 gradients are made by 3-fold dilution. Only the diluent PBS is added to the last well as a negative control, and incubated at 37°C for 1h. Wash five times with 300ul / well PBS-T solution, then add Anti-Human Fc-HRP secondary antibody diluted 1:40000 with 1% BSA-PBS solution and incubate at 37°C for 1h. Use 100ul / well of TMB color development kit to develop color, develop color at room temperature for 8min, and...

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Abstract

The invention relates to the field of biotechnology, in particular to an anti-EGFR (epidermal growth factor receptor) and anti-CD3 (cluster of differentiation 3) bispecific antibody and an application thereof. The bispecific antibody can be specifically bound with EGFR of tumor cell surface antigens and CD3, namely, CD3 molecules, of immune cell surface antigens; a single-chain antibody ScFv of an anti-CD3 antibody is located at the C terminal of a constant region of an anti-EGFR antibody. The invention further provides a preparation method of the bispecific antibody and a clinical application of the bispecific antibody. The bispecific antibody has higher affinity and is used for treating tumor diseases caused by high expression or abnormal expression of the EGFR and other diseases caused by EGFR overexpression.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing and preparing a bispecific antibody against EGFR and CD3, and the application of the antibody in diseases. Background technique [0002] Bispecific antibody (bispecific antibody, BiAb) is an artificial antibody composed of two different antibody fragments, which can specifically recognize and bind two different antigens or two different epitopes. [0003] Monoclonal antibodies are widely used in the treatment of cancer, inflammation, and other diseases. Because these antibodies are directed against a single target, many patients do not respond adequately to a single therapy, and drug resistance often occurs. Bispecific antibodies can recognize two different antigens or epitopes at the same time, and can be used as a medium to redirect immune effector cells, such as natural killer cells and T cells, to enhance the killing function of tumor cells. In addition...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/30C07K16/28C12N15/13C12N15/85A61K39/395A61P35/00
CPCC07K16/2809C07K16/30C07K16/468C07K2317/31C07K2317/52C07K2317/56C07K2317/622C07K2317/94C07K2317/92A61K2039/505C07K2317/64C07K2317/35C07K16/2863C07K2317/73A61P35/00C07K2317/14
Inventor 周海平张稳徐萌裴爽昝琰璐文圣梅白义白先宏
Owner BEIJING DONGFANG BIOTECH
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