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Human monoclonal antibody of human endophloeodal growth factor receptor 2 protein and its preparing process

An endothelial growth factor and monoclonal antibody technology, applied in the field of biomedicine, can solve problems such as the inability to fundamentally solve the HAMA problem, and achieve the effects of improved effect, long half-life and high yield

Inactive Publication Date: 2003-02-12
陕西超英生物医学研究开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the monoclonal antibody produced by the above technology can greatly reduce the HAMA response, it cannot fundamentally solve the HAMA problem because the mouse IgG CDR still exists in the monoclonal antibody molecule

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of fully human HER2 / neu monoclonal antibody by obtaining sensitized B cells from peripheral blood

[0040] Step ①, first determine the target volunteers, select 100 volunteers, all of whom are breast cancer patients, take 5ml of venous blood each, separate the serum, store at -20°C, and screen through the commercial HER2 ELISA KIT, the anti-HER2 antibody is positive and the titer Those whose ratio is greater than 1:64 are the target volunteers; then take 50ml of peripheral blood from the target volunteers, add lymphocyte stratification solution, centrifuge at 200×g, take the interface B cell layer, and take 100ul B cells for immune bead test to determine whether the B cells are sensitization. The sensitized B cells were combined and washed 3 times with RPMI1640. Step ②, use RPMI1640 to regulate B cells to 5×10 6 / ml, human myeloma cells adjusted to 2×10 for Karpas707H RPMI 6 / ml. Mix Karpas 707H with 1ml of sensitized human B cells, add 2ml of...

Embodiment 2

[0041] Example 2: Preparation of fully human HER2 / neu monoclonal antibody by obtaining sensitized B cells through in vitro immunization

[0042] Step ①, purchase 400ml of freshly collected venous blood anticoagulated with sodium citrate from the blood bank, separate the plasma, confirm that it is negative for anti-HER2 antibody by commercial HER2 ELISA KIT, add lymphocyte stratification solution, centrifuge at 200×g, and take the interface B cell layer. Take 100ul B cells for immunobead test to determine that the B cells are not sensitized. Unsensitized human peripheral blood B cells were cultured in conditioned medium for 24 hours, then transferred to complete medium for 5 days. The complete medium contained HER2 / neu extracellular region 2ng / ml, AB type human serum 1%, IL -2 100U / ml, IL-4 8ng / ml, IL-6 16ng / ml, macrophage 1×10 5 / ml, 28% of the B cells were sensitized as determined by the immunobead test, and the sensitized B cells were washed 3 times with RPMI1640; in step ...

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PUM

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Abstract

A humanized monoclonal antibody of human endophloeodal growth factor receptor 2 protein is prepared from enternal segment of HER2 / neucell through such steps as culturing the B cells of the person sensitive to HER2; fusing the said B cells with human osteoma cells (karpas 707 H); screening the external segment monoclonal anti body against HER2 cell; and amplification of the said monoclonal antibody. It features that antigen epitope is the external segment polypeptide of HER2 / neu cell for specifically binding the external segment of HER2 / neu cell, and the all gene sequences for syntehsis come from human B lymph cells and human osteoma cells.

Description

Technical field: [0001] The invention relates to the field of biomedicine, in particular to a fully human monoclonal antibody of human endothelial growth factor receptor 2 protein and a preparation method thereof. Background technique: [0002] Human endothelial growth factor receptor 2 protein (HER-2 / neu protein) is a single-chain transmembrane glycoprotein encoded by the proto-oncogene HER-2 / neu located on human chromosome 17 q11-22, with a molecular weight of 185kD. Therefore, it is also called P185. HER-2 / neu gene amplification or protein overexpression can often be detected in cells of many malignant tumors such as breast cancer, ovarian cancer, lung cancer, gastric cancer, and colon cancer, so the overexpression of HER-2 / neu protein is related to the occurrence of tumors. When the HER-2 / neu protein is overexpressed on the cell surface, the number of heterodimers formed by the HER-2 / neu receptor increases, and the cell signaling system will be overactivated, resulting ...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12P21/08
Inventor 刘镭李军王太重
Owner 陕西超英生物医学研究开发有限公司
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