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Anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein and application thereof

A technology of fusion protein and single-chain antibody, which is applied in the field of antibody protein, can solve the problems of low adaptability of antibody and indicator coupling, limited imaging effect, and single target, so as to broaden the application mode in vivo and reduce the false positive rate , the effect of improving the diagnosis rate

Active Publication Date: 2018-05-22
北京格根生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] With the continuous development of the existing technology, the demand for tumor imaging and treatment based on the specific recognition function of antibodies is increasing. However, the antibodies in the prior art also have poor tumor binding effects due to low specificity, and the target is single so that There is a risk of missed detection, the detection cost is increased due to the single labeling of the coupling agent, and the coupling adaptability of the antibody and the indicator is not high enough to limit the imaging effect, which limits the further application of the above technology

Method used

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  • Anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein and application thereof
  • Anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein and application thereof
  • Anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of anti-VEGF and anti-EGFR bispecific antibody

[0037] 1.1 Establishment of high-capacity natural antibody library.

[0038] Isolation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10ml of peripheral blood was extracted from each person. Dilute 1:1 with RPMI-1640 culture medium containing 10% heparin, add to the centrifuge tube containing lymphocyte separation medium (the volume ratio of diluted venous blood to lymphocyte separation medium is 2:1), 2,000×g, Centrifuge for 17 minutes. Aspirate the milky white mononuclear cell layer at the interface of the lymphocyte separation solution, and wash twice with PBS buffer.

[0039] 1.2 Extraction of total cellular RNA

[0040] Press every 5×10 6 Add Trizol reagent at the ratio of cells / ml, and lyse the cells by pipetting. Incubate at room temperature for 5 minutes, transfer to a DEPC-treated EP tube, add 1 / 5 volume of chloroform, shake vigorously ...

Embodiment 2

[0097] Example 2. Purification of bispecific antibodies

[0098] The sequences of the two ScFvs were re-genetically synthesized in the order that the heavy chain variable region and the light chain variable region of the VEGF single-chain antibody were connected to the light chain variable region and the heavy chain variable region of the EGFR single-chain antibody. Composition of new bispecific antibodies. Synthesized by Beijing Nuosai Biotechnology Co., Ltd.

[0099] Pick a single colony and inoculate it in 5ml LB medium, and cultivate overnight at 37°C with vigorous shaking.

[0100] Inoculate the above bacterial solution into a Erlenmeyer flask containing 400ml LB (antibiotics) at a ratio of 1:100, and incubate vigorously at 37°C for 2 hours.

[0101] The final concentration was 1 mmol / LIPTG, and the expression was induced at 37°C for 3-4 hours.

[0102] Collect the culture medium, centrifuge at 5,000 rpm for 10 minutes, and discard the supernatant. Wash the bacterial ...

Embodiment 3

[0109] Example 3. FITC-labeled VEGF ScFv, EGFR ScFv and VEGF ScFv+EGFR ScFv on the immunofluorescence results of pancreatic cancer tumor cell panc-1 (confocal)

[0110] The pancreatic cancer cell line panc-1 was used to verify the binding of VEGF ScFv, EGFR ScFv and VEGF ScFv+EGFR ScFv to tumor cells.

[0111] First, FITC (fluorescein isothiocyanate) or irdye800 labeled VEGF ScFv, EGFR ScFv and VEGFScFv+EGFR ScFv, the steps are as follows:

[0112] (1) Dialyze the protein to be cross-linked (concentration ≥ 1 mg / ml) against the cross-linking reaction solution three times (4° C.) until pH = 9.0. Preparation method of cross-linking reaction solution: 7.56g NaHCO 3 , 1.06g Na2CO 3 , 7.36g NaCl, add water to make up to 1L.

[0113] (2) FITC was dissolved in DMSO at a concentration of 1 mg / ml. The FITC used for each cross-linking should be freshly prepared and protected from light.

[0114] (3) Slowly add FITC to the antibody solution according to the ratio of P:F (protein: FI...

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Abstract

The invention discloses anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein. The anti-VEGF and anti-EGFR bispecific antibody fusion protein is prepared from an anti-VEGF single-chain antibody and an anti-EGFR single-chain antibody, has two groups of unique CDR region sequences which are capable of respectively identifying VEGF and EGFR, has high-specificity identification ability on VEGF and EGFR and has an excellent tumor binding effect and an excellent tumor locating effect. The invention also discloses application of the anti-VEGF and anti-EGFR bispecific antibody fusion protein coupling with an indicator in preparing an in-vitro tumor diagnosis reagent and an in-vivo bioimaging agent. In-vivo tumor imaging can be carried out by the anti-VEGF and anti-EGFR bispecific antibody fusion protein coupling with a MRI contrast medium and a fluorescent imaging agent; the molecular weight of the anti-VEGF and anti-EGFR bispecific antibody fusion protein is small and the fusion protein can be expressed in a prokaryotic cell expression system, and the production cost of antibody medicine is greatly reduced.

Description

technical field [0001] The invention discloses an antibody protein, more specifically, the invention discloses a bispecific antibody fusion protein. Background technique [0002] Tumor is the biggest disease that threatens human survival. The World Health Organization research shows that in 2012, the number of cancer cases in China was 3.065 million, accounting for about one-fifth of the global incidence; the number of cancer deaths was 2.205 million, accounting for about one-quarter of the global cancer deaths. [0003] The clinical diagnosis technology of tumor is also improving gradually. From the initial detection of tumor markers, ultrasound, fluoroscopy, etc., to the detection of more protein and molecular levels of tumor-related factors, CT, nuclear magnetic resonance, PET, SPET, and minimally invasive biopsy of tumors, etc. have been widely used clinically. [0004] Tumor diagnosis is divided into in vitro and in vivo diagnosis. In vitro, immunohistochemical stai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K49/00A61K49/16A61K49/08
CPCA61K49/0002A61K49/0058A61K49/085A61K49/16C07K16/22C07K16/2863C07K2317/31C07K2317/565C07K2317/622
Inventor 谢珞琨满来李峥许春萍
Owner 北京格根生物科技有限公司
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