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Bispecific antibody capable of resisting B cell lymphoma and application thereof

A bispecific antibody, lymphoma technology, applied in the field of genetic engineering and protein engineering, can solve the problems of insufficient tumor killing efficiency, difficult enrichment of T lymphocytes, lack of targeted tumor cells, etc., to improve utilization efficiency and therapeutic effect , The effect of avoiding the occurrence of HAMA and reducing the phenomenon of drug resistance

Inactive Publication Date: 2014-05-14
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of the function of targeting tumor cells, it is difficult for activated T lymphocytes to effectively enrich the tumor site, resulting in insufficient tumor killing efficiency.

Method used

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  • Bispecific antibody capable of resisting B cell lymphoma and application thereof
  • Bispecific antibody capable of resisting B cell lymphoma and application thereof
  • Bispecific antibody capable of resisting B cell lymphoma and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Cloning of DNA sequences encoding bispecific antibodies and construction of recombinant vectors

[0063] The gene fragment encoding the bispecific antibody in the present invention can be obtained by classical molecular biology techniques, and the gene sequence can be optimized for the mammalian expression system in order to obtain better expression. Recombinant vectors can be obtained by reconnecting bispecific antibody gene fragments with corresponding expression vectors, which are suitable for expression and screening of mammalian cells.

[0064] 1. Obtain the gene fragment encoding CD19-CD3 bispecific antibody

[0065] The variable region gene of the human CD19 antibody and the variable region gene of the human CD3 antibody in the present invention are respectively obtained by screening the whole human antibody library (the construction method of the whole human antibody library can be found in E, et al. Recombining germline-derived CDR sequences for cr...

Embodiment 2

[0068] Example 2 Expression and purification of the bispecific antibody of the present invention in cells

[0069] The bispecific antibody in the present invention is expressed in CHO cells and secreted into the culture medium, and purified by nickel column affinity chromatography.

[0070] 1. Transient expression of bispecific antibody in CHO cells

[0071] After obtaining high-purity recombinant plasmids encoding bispecific antibodies, use Lipofectamine 2000 Plasmid Transfection Kit (Invitrogen) to transfect CHO cells (ATCC) with the recombinant plasmids, and collect CHO cell supernatants after culturing in serum-free medium for three days solution, the expression of bispecific antibodies can be detected by Western blotting (see Figure 4 ). This method can be used to quickly obtain a small amount of bispecific antibody protein, and its concentration can be quantitatively detected by ELISA, and the primary antibody used can be anti-His6 or Flag antibody.

[0072] 2. Stabl...

Embodiment 3

[0076] Example 3 In vitro binding experiment of the bispecific antibody of the present invention and cells

[0077] The bispecific antibody of the present invention may be able to bind the corresponding target cells in vitro. In the present invention, Raji cells are used as CD19-positive cells, Jurkat cells are used as CD3-positive cells, and the bispecific antibody of the present invention is used to detect their cell-binding activity.

[0078] 1. Detection of binding activity of bispecific antibody to Raji cells by flow cytometry

[0079] The bispecific antibody was mixed with Raji cells in 1% bovine serum albumin (BSA) and 0.02% sodium azide in PBS (staining buffer), and incubated on ice for 1 hour. Cells were washed twice with PBS buffer, and then incubated with mouse anti-His6 antibody on ice for 1 hour. After the cells were washed with PBS buffer, they were incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse antibody in staining buffer for 30 min...

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Abstract

The invention relates to the technical fields of genetic engineering and protein engineering, in particular relates to DNA (deoxyribonucleic acid) for encoding recombinant fusion protein containing human CD19 antibody variable region and human CD3 antibody variable region fragments, fusion protein encoded by DNA, a production method of the fusion protein, pharmaceutical application of the fusion protein and a treatment method using the fusion protein. The invention provides bispecific antibody protein containing human CD19scFv and CD3scFv. The bispecific antibody protein can be combined with positive CD19 and CD3 positive cells, has good bioactivities in vivo and vitro, can activate human T lymphocyte, kill B lymphoma cells, and has good application prospects.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and protein engineering, in particular, to a DNA encoding a recombinant fusion protein comprising a variable region of a human CD19 antibody and a fragment of a variable region of a human CD3 antibody, the encoded fusion protein, the fusion The production method of the protein, the pharmaceutical use of the fusion protein and the treatment method of the fusion protein. Background of the invention [0002] B-cell lymphoma is a malignant tumor with a high incidence rate, which seriously endangers human health. At present, its antibody drug therapy has been affirmed, and antibody drugs such as Rituximab have been marketed. Antibody drug targets for B-cell lymphoma are mainly CD molecules with increased specific expression in B-lymphoma cells, such as CD19, CD20, and CD22. Antibody drugs targeting these CD molecules can induce apoptosis of B lymphoma cells, thereby achieving the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/395A61P35/02
Inventor 杨金亮勾蓝图杨莉魏于全
Owner SICHUAN UNIV
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