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Human antibody sample molecule TEM8-Fc based on tumour endocytosis sign 8 and its application in tumour treatment

A technology of endothelial cells and human antibodies, applied in the direction of anti-tumor drugs, medical preparations containing active ingredients, cells modified by introducing foreign genetic materials, etc., to achieve wide indications, small side effects, and large market effects

Inactive Publication Date: 2008-01-30
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the development of TEM8-Fc fusion protein for tumor treatment at home and abroad

Method used

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  • Human antibody sample molecule TEM8-Fc based on tumour endocytosis sign 8 and its application in tumour treatment
  • Human antibody sample molecule TEM8-Fc based on tumour endocytosis sign 8 and its application in tumour treatment
  • Human antibody sample molecule TEM8-Fc based on tumour endocytosis sign 8 and its application in tumour treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. SDS-PAGE analysis of gene recombination products

[0035] According to the method described in Chinese patent 200510084233.5, gene recombinant TEM8-Fc fusion protein was expressed in rCHO cells, and rCHO cells were cultivated with serum-free medium (Chinese patent ZL 200310124257.X), and nProtein A Sepharose 4 Fast Flow affinity column ( Amersham Biosciences) purified TEM8-Fc from the cell culture supernatant. The schematic diagram of the molecular structure of the TEM8-Fc fusion protein is shown in FIG. 1 . If TEM8-Fc can be correctly expressed in CHO cells, the expressed protein is expected to be a homodimeric antibody-like molecule, that is, the single chain of TEM8-Fc is connected into a dimer by an interchain disulfide bond at the hinge region (Figure 1 ). The single-chain TEM8-Fc molecule has a total of 459 amino acid residues, of which the first 27 amino acids are the signal peptide of TEM8, which are excised when extracellularly secreted into the ce...

Embodiment 2

[0037] Example 2. ELISA confirmed the specific binding of TEM8 to uPA or tPA

[0038] Gene recombination of prourokinase (Pro-UK, source: Hu Xianwen, Xiao Chengzu, Li Zuohu. Pilotproduction of u-PA with porous microcarrier cell culture. Cytotechnology.2000.33(1~3): 13-19), low gene recombination Molecular weight urokinase (LMW-UK, Chinese patent: low molecular weight urokinase mutant and its expression vector, application number: 200610065813.4, publication number CN1880449) and gene recombinant tissue plasminogen activator (tPA, source: Roche company) Coat NUNCTM enzyme-linked plate at 0.8 μg / well, add TEM8-Fc fusion protein after blocking with 3% BSA, fully wash after incubation, then add HRP-labeled goat anti-human IgG (H+L) enzyme-labeled polyclonal antibody, add after incubation TMB reagent color development, OD value measured at 405nm. The humanized monoclonal antibody Herceptin (Roche) was used as a negative control. The results are shown in Figure 3. For the first ti...

Embodiment 3

[0039] Example 3. TEM8-Fc fusion protein can inhibit the migration of tumor cells induced by uPA

[0040] Since uPA plays a pivotal role in the migration of tumor cells, blocking uPA may inhibit the migration of tumor cells. The overexpression of TEM8 on the surface of liver cancer cells HepG2 (ATCC No.HB8065) ​​was analyzed by flow cytometry (Fig. 4(a)). The anti-TEM8 rabbit polyclonal antibody used in this experiment was purchased from Santa Cruz Company. Transwell in vitro cell migration assay (source: Corning Costar Corporation, http: / / www.tc.umn.edu / ~shimi002 / SOPtranswell.pdf) was used to verify that TEM8-Fc could block uPA to inhibit the migration of HepG2 tumor cells.

[0041] Method: HepG2 cells were divided into 1×10 per well 5 Two cells were inoculated into the upper chamber of Transwells, HepG2 cells were treated with LMW-UK (10nmol / L) for 4 hours to stimulate migration, and the blocking effect of TEM8-Fc (25g / mL) on LMW-UK was observed. After 4 hours of exposure,...

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Abstract

The invention discloses a human antibody sample molecular TEM8-Fc based on tumor endothelium marker 8 (TEM8) and the application thereof in tumor therapy and pertains to the biological pharmaceutical field. TEM8-Fc fusion protein is recombinant dimer fusion protein by the linkage of the 1-227 amino acid at extracellular domain of the TEM8, fc fragment, heavy chain constant region 2 and heavy chain constant region 3 of human immunoglobulin IgG1; mature single strand TEM8-Fc has 432 amino acid residues and the dimer has 864 amino acid residues. The invention has the advantages of small side effect, high specificity and wide indication. Animal experiments show that TEM8-Fc can not only effectively inhibit growth of tumor cells such as breast cancer, colorectal cancer, liver cancer, etc. in nude mice, but also can inhibit metastasis of cancer cells such as breast cancer, colorectal cancer, liver cancer, etc., and the angiogenesis in tumor tissues and reverse tumor cells phenotype.

Description

technical field [0001] The invention relates to a human antibody-like molecule TEM8-Fc based on tumor endothelial cell marker 8 (TEM8) and its application in tumor treatment, belonging to the field of biopharmaceuticals. Background technique [0002] Angiogenesis and tumor cell metastasis are inevitable processes in the occurrence and development of all advanced malignant solid tumors. The key molecules related to angiogenesis and tumor cell metastasis naturally become ideal targets for the development of tumor therapeutic drugs, and the development of drugs that antagonize or block the pathways of angiogenesis and tumor cell invasion and metastasis should have broad-spectrum anti-tumor activity. [0003] Deregulation of angiogenesis is a major contributor to malignant tumor growth and metastasis. Therefore, the anti-tumor strategy of "starving the tumor" by inhibiting the growth of tumor blood vessels has aroused great interest and widespread concern of tumor researchers a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61P35/00C12N15/62C12N15/79C12N5/10
Inventor 胡显文高丽华段海峰陈惠鹏郗永义陈金龙
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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