Preparation of bispecific antibody targeting human BCMA and activating NK cells and application of bispecific antibody

A bispecific antibody and NK cell technology, applied in the fields of application, antibody, and specific peptide, can solve the problems of immune escape and inability to activate immune cells, and achieve obvious effects, high affinity, and obvious cell growth effects

Active Publication Date: 2020-06-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes an anti cancer agent called BCA-3 that targets specific parts (cell) within blood vessels or lymphatic fluid around certain types of cancers. It has been found effective at killing these areas without causing harmful side reactions like chemotherapy drugs used against breast cancer patients.

Problems solved by technology

Technological Problem: Current Anti-BCMA Therapies Target CD19+CD20+myelocyte stem/progenitor white blood cells called monocular spleglicy syndrome or multifocal leukemia are associated with poor outcomes due to reduced efficacy against cancer development. These treatments aim at killing these abnormal cells without affecting healthy ones like hormones. They rely heavily on specific interactions between certain parts of DNA and various components involved in inflammatory responses. By combining them together they enhances the efficiency of chemoimmunocompetence and immunesystem functions.

Method used

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  • Preparation of bispecific antibody targeting human BCMA and activating NK cells and application of bispecific antibody
  • Preparation of bispecific antibody targeting human BCMA and activating NK cells and application of bispecific antibody
  • Preparation of bispecific antibody targeting human BCMA and activating NK cells and application of bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of bispecific antibody 2A9-MICA

[0038] Using overlap PCR technology through flexible peptide G 4 S was used for gene splicing and enzyme cleavage sites were added at both ends. The PCR product was detected by 1% agarose gel electrophoresis, and the target gene fragment was recovered with the agarose gel recovery kit. The target gene and pCMV3 were double-digested separately, and the target gene and plasmid fragments were recovered after digestion. T4 ligase was ligated overnight at 16°C to obtain recombinant plasmid pCMV3-2A9 MICA. CaCl 2 The ligation product was transformed into Escherichia coli DH5α for amplification and storage. The result is as figure 1 As shown in A.

Embodiment 2

[0039] Example 2: Expression and purification of bispecific antibody 2A9-MICA

[0040] First, the recombinant plasmid pCMV3-2A9 MICA was transfected into HEK293 cells with PEI transfection reagent, the medium was changed the next day and the cells were passaged, and the scale of cell fermentation culture was gradually expanded, the cell culture medium was collected, and the samples were filtered through a 0.22 μm filter membrane Purify by Histrap affinity chromatography, and finally obtain a large amount of the target protein. The expressed 2A9-MICA protein structure mimics as figure 1 Shown in B. The detailed steps of purification are as follows: first wash the Histrap column with double distilled water for 10 minutes, then equilibrate the column with BindingBuffer for 10 minutes, and the flow rate of washing is 1ml / min. Next, put the crude single-chain antibody protein solution on the column, and control the flow rate to be half of the washing flow rate. Collect the efflu...

Embodiment 3

[0041] Example 3: Detection of binding ability of 2A9-MICA to various tumor cell lines by flow cytometry

[0042] In this experiment, 2×10 5 NCI-H929, OPM-2, RPMI-8226 and Raji cells were resuspended in 250 μl PBS to prepare single cell suspension. Then, an equal volume concentration of 500 nM 2A9-MICA was added to each cell suspension for co-incubation for 1 h. After washing with PBS to remove unbound rG7S-MICA, the tumor cells were incubated with FITC-labeled anti-his mAb. After washing with PBS, the fusion protein bound to the cell surface was detected by flow cytometry, and the 2A9-MICA sensitive type was screened. Tumor cell lines. The result is as figure 2 shown in A-2D.

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Abstract

The invention discloses a bispecific antibody targeting human BCMA and activating NK cells and application of the bispecific antibody, and belongs to the technical field of genetic engineering antibodies. An anti-human BCMA single chain antibody 2A9 is connected to extracellular 1-3 regions of a MICA molecule through G4S flexible peptide by using a genetic engineering technology, and a mammalian eukaryotic expression system expresses 2A9-MICA. The invention further provides a method for expressing and purifying the 2A9-MICA bispecific antibody, secretory expressing is carried out by HEK293 cells, and target protein is obtained through affinity chromatography purification. The bispecific antibody 2A9-MICA can specifically bind to a BCMA molecule on the surface of a multiple myeloma cell andan activated receptor NKG2D on the surface of an NK cell simultaneously, the immune monitoring function of the NKG2D pathway-induced NK cell on a BCMA positive myeloma cell is remodeled, and the purpose of activating a body immune system to effectively kill the BCMA-positive myeloma cell is finally achieved.

Description

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Claims

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Application Information

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Owner CHINA PHARM UNIV
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